Methods and compositions for prime editing nucleotide sequences

ABSTRACT

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

RELATED APPLICATIONS

This application is a continuation of and claims priority under 35U.S.C. §§ 120 and 365(c) to U.S. patent application U.S. Ser. No.18/064,738, filed on Dec. 12, 2022, which is a continuation of andclaims under 35 U.S.C. § 120 to U.S. patent application U.S. Ser. No.17/219,635, filed on Mar. 31, 2021, which claims priority under 35U.S.C. §§ 120 and 365(c) to and is a continuation of International PCTApplication PCT/US2020/023730, filed on Mar. 19, 2020, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.62/820,813, filed on Mar. 19, 2019, U.S. Provisional Application No.62/858,958, filed on Jun. 7, 2019, U.S. Provisional Application No.62/889,996, filed on Aug. 21, 2019, U.S. Provisional Application No.62/922,654, filed on Aug. 21, 2019, U.S. Provisional Application No.62/913,553, filed on Oct. 10, 2019, U.S. Provisional Application No.62/973,558, filed on Oct. 10, 2019, U.S. Provisional Application No.62/931,195, filed on Nov. 5, 2019, U.S. Provisional Application No.62/944,231, filed on Dec. 5, 2019, U.S. Provisional Application No.62/974,537, filed on Dec. 5, 2019, U.S. Provisional Application No.62/991,069, filed on Mar. 17, 2020, and U.S. Provisional Application No.63/100,548, filed on Mar. 17, 2020, the entire contents of each of whichis incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NumbersAI142756, EB022376, GM007726, GM118062, GM954507, and HG009490 awardedby the National Institutes of Health. The government has certain rightsin the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE

The contents of the electronic sequence listing(B119570096US05-SEQ-TNG.xml; Size: 5,438,324 bytes; and Date ofCreation: May 31, 2023) is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Pathogenic single nucleotide mutations contribute to approximately 50%of human diseases for which there is a genetic component,⁷ according tosome estimates. Unfortunately, treatment options for patients with thesegenetic disorders remain extremely limited, despite decades of genetherapy exploration⁸. Perhaps the most parsimonious solution to thistherapeutic challenge is direct correction of single nucleotidemutations in patient genomes, which would address the root cause ofdisease and would likely provide lasting benefit. Although such astrategy was previously unthinkable, recent improvements in genomeediting capabilities brought about by the advent of the CRISPR/Cassystem⁹ have now brought this therapeutic approach within reach. Bystraightforward design of a guide RNA (gRNA) sequence that contains ˜20nucleotides complementary to the target DNA sequence, nearly anyconceivable genomic site can be specifically accessed by CRISPRassociated (Cas) nucleases^(1,2). To date, several monomeric bacterialCas nuclease systems have been identified and adapted for genome editingapplications¹⁰. This natural diversity of Cas nucleases, along with agrowing collection of engineered variants¹¹⁻¹⁴, offers fertile groundfor developing new genome editing technologies.

While gene disruption with CRISPR is now a mature technique, precisionediting of single base pairs in the human genome remains a majorchallenge³. Homology directed repair (HDR) has long been used in humancells and other organisms to insert, correct, or exchange DNA sequencesat sites of double strand breaks (DSBs) using donor DNA repair templatesthat encode the desired edits¹⁵. However, traditional HDR has very lowefficiency in most human cell types, particularly in non-dividing cells,and competing non-homologous end joining (NHEJ) leads predominantly toinsertion-deletion (indel) byproducts¹⁶. Other issues relate to thegeneration of DSBs, which can give rise to large chromosomalrearrangements and deletions at target loci¹⁷, or activate the p53 axisleading to growth arrest and apoptosis^(18,19).

Several approaches have been explored to address these drawbacks of HDR.For example, repair of single-stranded DNA breaks (nicks) witholigonucleotide donors has been shown to reduce indel formation, butyields of desired repair products remain low²⁰. Other strategies attemptto bias repair toward HDR over NHEJ using small molecule and biologicreagents²¹⁻²³. However, the effectiveness of these methods is likelycell-type dependent, and perturbation of the normal cell state couldlead to undesirable and unforeseeable effects.

Recently, the inventors, led by Prof. David Liu et al., developed baseediting as a technology that edits target nucleotides without creatingDSBs or relying on HDR^(4-6,24-27). Direct modification of DNA bases byCas-fused deaminase enzymes allows for C·G to T·A, or A·T to G·C, basepair conversions in a short target window (˜5-7 bases) with very highefficiency. As a result, base editors have been rapidly adopted by thescientific community. However, the following factors limit theirgenerality for precision genome editing: (1) “bystander editing” ofnon-target C or A bases within the target window are observed; (2)target nucleotide product mixtures are observed; (3) target bases mustbe located 15±2 nucleotides upstream of a PAM sequence; and (5) repairof small insertion and deletion mutations is not possible.

Therefore, the development of programmable editors that are flexiblycapable of introducing any desired single nucleotide change and/or whichcould install base pair insertions or deletions (e.g., at least 1, 2, 3,4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50,60, 70, 80, 90, 100, or more base pair insertions or deletions) and/orwhich could alter or modify the nucleotide sequence at a target sitewith high specificity and efficiency would substantially expand thescope and therapeutic potential of genome editing technologies based onCRISPR.

SUMMARY OF THE INVENTION

The present invention describes an entirely new platform for genomeediting called “prime editing.” Prime editing is a versatile and precisegenome editing method that directly writes new genetic information intoa specified DNA site using a nucleic acid programmable DNA bindingprotein (“napDNAbp”) working in association with a polymerase (i.e., inthe form of a fusion protein or otherwise provided in trans with thenapDNAbp), wherein the prime editing system is programmed with a primeediting (PE) guide RNA (“PEgRNA”) that both specifies the target siteand templates the synthesis of the desired edit in the form of areplacement DNA strand by way of an extension (either DNA or RNA)engineered onto a guide RNA (e.g., at the 5′ or 3′ end, or at aninternal portion of a guide RNA). The replacement strand containing thedesired edit (e.g., a single nucleobase substitution) shares the samesequence as the endogenous strand of the target site to be edited (withthe exception that it includes the desired edit). Through DNA repairand/or replication machinery, the endogenous strand of the target siteis replaced by the newly synthesized replacement strand containing thedesired edit. In some cases, prime editing may be thought of as a“search-and-replace” genome editing technology since the prime editors,as described herein, not only search and locate the desired target siteto be edited, but at the same time, encode a replacement strandcontaining a desired edit which is installed in place of thecorresponding target site endogenous DNA strand.

The prime editors of the present disclosure relate, in part, to thediscovery that the mechanism of target-primed reverse transcription(TPRT) or “prime editing” can be leveraged or adapted for conductingprecision CRISPR/Cas-based genome editing with high efficiency andgenetic flexibility (e.g., as depicted in various embodiments of FIGS.1A-1F). TPRT is naturally used by mobile DNA elements, such as mammaliannon-LTR retrotransposons and bacterial Group II introns^(28,29). Theinventors have herein used Cas protein-reverse transcriptase fusions orrelated systems to target a specific DNA sequence with a guide RNA,generate a single strand nick at the target site, and use the nicked DNAas a primer for reverse transcription of an engineered reversetranscriptase template that is integrated with the guide RNA. However,while the concept begins with prime editors that use reversetranscriptases as the DNA polymerase component, the prime editorsdescribed herein are not limited to reverse transcriptases but mayinclude the use of virtually and DNA polymerase. Indeed, while theapplication throughout may refer to prime editors with “reversetranscriptases,” it is set forth here that reverse transcriptases areonly one type of DNA polymerase that may work with prime editing. Thus,wherever the specification mentions “reverse transcriptases,” the personhaving ordinary skill in the art should appreciate that any suitable DNApolymerase may be used in place of the reverse transcriptase. Thus, inone aspect, the prime editors may comprise Cas9 (or an equivalentnapDNAbp) which is programmed to target a DNA sequence by associating itwith a specialized guide RNA (i.e., PEgRNA) containing a spacer sequencethat anneals to a complementary protospacer in the target DNA. Thespecialized guide RNA also contains new genetic information in the formof an extension that encodes a replacement strand of DNA containing adesired genetic alteration which is used to replace a correspondingendogenous DNA strand at the target site. To transfer information fromthe PEgRNA to the target DNA, the mechanism of prime editing involvesnicking the target site in one strand of the DNA to expose a 3′-hydroxylgroup. The exposed 3′-hydroxyl group can then be used to prime the DNApolymerization of the edit-encoding extension on PEgRNA directly intothe target site. In various embodiments, the extension—which providesthe template for polymerization of the replacement strand containing theedit—can be formed from RNA or DNA. In the case of an RNA extension, thepolymerase of the prime editor can be an RNA-dependent DNA polymerase(such as, a reverse transcriptase). In the case of a DNA extension, thepolymerase of the prime editor may be a DNA-dependent DNA polymerase.

The newly synthesized strand (i.e., the replacement DNA strandcontaining the desired edit) that is formed by the herein disclosedprime editors would be homologous to the genomic target sequence (i.e.,have the same sequence as) except for the inclusion of a desirednucleotide change (e.g., a single nucleotide change, a deletion, or aninsertion, or a combination thereof). The newly synthesized (orreplacement) strand of DNA may also be referred to as a single strandDNA flap, which would compete for hybridization with the complementaryhomologous endogenous DNA strand, thereby displacing the correspondingendogenous strand. In certain embodiments, the system can be combinedwith the use of an error-prone reverse transcriptase enzyme (e.g.,provided as a fusion protein with the Cas9 domain, or provided in transto the Cas9 domain). The error-prone reverse transcriptase enzyme canintroduce alterations during synthesis of the single strand DNA flap.Thus, in certain embodiments, error-prone reverse transcriptase can beutilized to introduce nucleotide changes to the target DNA. Depending onthe error-prone reverse transcriptase that is used with the system, thechanges can be random or non-random.

Resolution of the hybridized intermediate (comprising the single strandDNA flap synthesized by the reverse transcriptase hybridized to theendogenous DNA strand) can include removal of the resulting displacedflap of endogenous DNA (e.g., with a 5′ end DNA flap endonuclease,FEN1), ligation of the synthesized single strand DNA flap to the targetDNA, and assimilation of the desired nucleotide change as a result ofcellular DNA repair and/or replication processes. Because templated DNAsynthesis offers single nucleotide precision for the modification of anynucleotide, including insertions and deletions, the scope of thisapproach is very broad and could foreseeably be used for myriadapplications in basic science and therapeutics.

In one aspect, the specification provides a fusion protein comprising anucleic acid programmable DNA binding protein (napDNAbp) and a reversetranscriptase. In various embodiments, the fusion protein is capable ofcarrying out genome editing by target-primed reverse transcription inthe presence of an extended guide RNA.

In certain embodiments, the napDNAbp has a nickase activity. ThenapDNAbp may also be a Cas9 protein or functional equivalent thereof,such as a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or aCas9 nickase (nCas9).

In certain embodiments, the napDNAbp is selected from the groupconsisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c,and Argonaute and optionally has a nickase activity.

In other embodiments, the fusion protein when complexed with an extendedguide RNA is capable of binding to a target DNA sequence.

In still other embodiments, the target DNA sequence comprises a targetstrand and a complementary non-target strand.

In other embodiments, the binding of the fusion protein complexed to theextended guide RNA forms an R-loop. The R-loop can comprise (i) anRNA-DNA hybrid comprising the extended guide RNA and the target strand,and (ii) the complementary non-target strand.

In still other embodiments, the complementary non-target strand isnicked to form a reverse transcriptase priming sequence having a free 3′end.

In various embodiments, the extended guide RNA comprises (a) a guide RNAand (b) an RNA extension at the 5′ or the 3′ end of the guide RNA, or atan intramolecular location in the guide RNA. The RNA extension cancomprise (i) a reverse transcription template sequence comprising adesired nucleotide change, (ii) a reverse transcription primer bindingsite, and (iii) optionally, a linker sequence. In various embodiments,the reverse transcription template sequence may encode a single-strandDNA flap that is complementary to an endogenous DNA sequence adjacent tothe nick site, wherein the single-strand DNA flap comprises the desirednucleotide change.

In various embodiments, the RNA extension is at least 5 nucleotides, atleast 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, atleast 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides,at least 12 nucleotides, at least 13 nucleotides, at least 14nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, atleast 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides,at least 23 nucleotides, at least 24 nucleotides, or at least 25nucleotides in length.

In still other embodiments, the single-strand DNA flap may hybridize tothe endogenous DNA sequence adjacent to the nick site, therebyinstalling the desired nucleotide change. In still other embodiments,the single-stranded DNA flap displaces the endogenous DNA sequenceadjacent to the nick site and which has a free 5′ end. In certainembodiments, the displaced endogenous DNA having the 5′ end is excisedby the cell.

In various embodiments, the cellular repair of the single-strand DNAflap results in installation of the desired nucleotide change, therebyforming a desired product.

In various other embodiments, the desired nucleotide change is installedin an editing window that is between about −4 to +10 of the PAMsequence.

In still other embodiments, the desired nucleotide change is installedin an editing window that is between about −5 to +5 of the nick site, orbetween about −10 to +10 of the nick site, or between about −20 to +20of the nick site, or between about −30 to +30 of the nick site, orbetween about −40 to +40 of the nick site, or between about −50 to +50of the nick site, or between about −60 to +60 of the nick site, orbetween about −70 to +70 of the nick site, or between about −80 to +80of the nick site, or between about −90 to +90 of the nick site, orbetween about −100 to +100 of the nick site, or between about −200 to+200 of the nick site.

In various embodiments, the napDNAbp comprises an amino acid sequence ofSEQ ID NO: 18. In various other embodiments, the napDNAbp comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NOs: 26-39,42-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487 (Cas9); (SpCas9); SEQ ID NO: 77-86 (CP-Cas9); SEQ ID NO: 18-25and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12)

In other embodiments, the reverse transcriptase of the disclosed fusionproteins and/or compositions may comprise any one of the amino acidsequences of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149,154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766. In stillother embodiments, the reverse transcriptase may comprise an amino acidsequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical tothe amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122,128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716,739-742, and 766. These sequences may be naturally occurring reversetranscriptase sequences, e.g., from a retrovirus or a retrotransposon,of the sequences may be recombinant.

In various other embodiments, the fusion proteins herein disclosed maycomprise various structural configurations. For example, the fusionproteins may comprise the structure NH₂-[napDNAbp]-[reversetranscriptase]-COOH; or NH₂-[reverse transcriptase]-[napDNAbp]-COOH,wherein each instance of “]-[” indicates the presence of an optionallinker sequence.

In various embodiments, the linker sequence comprises an amino acidsequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769, or an aminoacid sequence that this at least 80%, 85%, or 90%, or 95%, or 99%identical to any one of the linker amino acid sequence of SEQ ID NOs:127, 165-176, 446, 453, and 767-769.

In various embodiments, the desired nucleotide change that isincorporated into the target DNA can be a single nucleotide change(e.g., a transition or transversion), an insertion of one or morenucleotides, or a deletion of one or more nucleotides.

In certain cases, the insertion is at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 200, at least 300, atleast 400, or at least 500 nucleotides in length.

In certain other cases, the deletion is at least 1, at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 200, at least 300, atleast 400, or at least 500 nucleotides in length.

In another aspect, the present disclosure provides an extended guide RNAcomprising a guide RNA and at least one RNA extension. The RNA extensioncan be positioned at the 3′ end of the guide RNA. In other embodiments,the RNA extension can be positioned at the 5′ of the guide RNA. In stillother embodiments, the RNA extension can be positioned at anintramolecular position within the guide RNA, however, preferable, theintramolecular positioning of the extended portion does not disrupt thefunctioning of the protospacer.

In various embodiments, the extended guide RNA is capable of binding toa napDNAbp and directing the napDNAbp to a target DNA sequence. Thetarget DNA sequence can comprise a target strand and a complementarynon-target strand, wherein the guide RNA hybridizes to the target strandto form an RNA-DNA hybrid and an R-loop.

In various embodiments of the extended guide RNA, the at least one RNAextension can comprise a reverse transcription template sequence. Invarious other embodiment, the RNA extension may further comprises areverse transcription primer binding site. In still further embodiments,the RNA extension may comprise a linker or spacer sequence that joinsthe RNA extension to the guide RNA.

In various embodiments, the RNA extension can be at least 5 nucleotides,at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides,at least 9 nucleotides, at least 10 nucleotides, at least 11nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, atleast 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides,at least 20 nucleotides, at least 21 nucleotides, at least 22nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, atleast 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides,at least 80 nucleotides, at least 90 nucleotides, at least 100nucleotides, at least 150 nucleotides, at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, or at least 500nucleotides in length.

In other embodiments, the reverse transcription template sequence is atleast 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, atleast 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, atleast 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides,at least 12 nucleotides, at least 13 nucleotides, at least 14nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, atleast 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides,at least 50 nucleotides, at least 60 nucleotides, at least 70nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, or at least 500 nucleotides in length.

In still other embodiments, wherein the reverse transcription primerbinding site sequence is at least 3 nucleotides, at least 4 nucleotides,at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides,at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides,at least 11 nucleotides, at least 12 nucleotides, at least 13nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, atleast 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides,at least 40 nucleotides, at least 50 nucleotides, at least 60nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, or at least 500nucleotides in length.

In other embodiments, the optional linker or spacer sequence is at least3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, atleast 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides,at least 15 nucleotides, at least 16 nucleotides, at least 17nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, atleast 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides,at least 80 nucleotides, at least 90 nucleotides, at least 100nucleotides, at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, or at least 500 nucleotides in length.

In various embodiments of the extended guide RNAs, the reversetranscription template sequence may encode a single-strand DNA flap thatis complementary to an endogenous DNA sequence adjacent to a nick site,wherein the single-strand DNA flap comprises a desired nucleotidechange. The single-stranded DNA flap may displace an endogenoussingle-strand DNA at the nick site. The displaced endogenoussingle-strand DNA at the nick site can have a 5′ end and form anendogenous flap, which can be excised by the cell. In variousembodiments, excision of the 5′ end endogenous flap can help driveproduct formation since removing the 5′ end endogenous flap encourageshybridization of the single-strand 3′ DNA flap to the correspondingcomplementary DNA strand, and the incorporation or assimilation of thedesired nucleotide change carried by the single-strand 3′ DNA flap intothe target DNA.

In various embodiments of the extended guide RNAs, the cellular repairof the single-strand DNA flap results in installation of the desirednucleotide change, thereby forming a desired product.

In certain embodiments, the PEgRNA comprises the nucleotide sequence ofSEQ ID NOs: 101-104, 131, 181-183, 222-234, 237-244, 277, 324-330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736,738, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493,3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911,3913-3929, and 3972-3989, or a nucleotide sequence having at least 85%,or at least 90%, or at least 95%, or at least 98%, or at least 99%sequence identity with any one of SEQ ID NOs: 101-104, 181-183, 223-234,237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505,641-649, 678-692, 735-736, 757-761, 776-777, 2997-3103, 3113-3121,3305-3455, 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874,3890-3901, 3905-3911, 3913-3929, and 3972-3989.

In yet another aspect of the invention, the specification provides forcomplexes comprising a fusion protein described herein and any extendedguide RNA described above.

In still other aspects of the invention, the specification provides acomplex comprising a napDNAbp and an extended guide RNA. The napDNAbpcan be a Cas9 nickase, or can be an amino acid sequence of SEQ ID NOs:42-57 (Cas9 nickase) and 65 (AsCas12a nickase), or an amino acidsequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical tothe amino acid sequence of any one of SEQ ID NOs: 42-57 (Cas9 nickase)and 65 (AsCas12a nickase).

In various embodiments involving a complex, the extended guide RNA iscapable of directing the napDNAbp to a target DNA sequence. In variousembodiments, a reverse transcriptase may be provided in trans, i.e.,provided from a different source than the complex itself. For example, areverse transcriptase could be provided to the same cell having thecomplex by introducing a separate vector separately encoding the reversetranscriptase.

In yet another aspect, the specification provides polynucleotides. Incertain embodiments, the polynucleotides may encode any of the fusionproteins disclosed herein. In certain other embodiments, thepolynucleotides may encode any of the napDNAbps disclosed herein. Instill further embodiments, the polynucleotides may encode any of thereverse transcriptases disclosed herein. In yet other embodiments, thepolynucleotides may encode any of the extended guide RNAs disclosedherein, any of the reverse transcription template sequences, or any ofthe reverse transcription primer sites, or any of the optional linkersequences.

In still other aspects, the specification provides vectors comprisingthe polynucleotides described herein. Thus, in certain embodiments, thevectors comprise polynucleotides for encoding the fusion proteinscomprising a napDNAbp and a reverse transcriptase. In other embodiments,the vectors comprise polynucleotides that separately encode a napDNAbpand reverse transcriptase. In still other embodiments, the vectors maycomprise polynucleotides that encode the extended guide RNAs. In variousembodiments, the vectors may comprise one or more polynucleotides thatencode napDNAbps, reverse transcriptase, and extended guide RNAs on thesame or separate vectors.

In still other aspects, the specification provides cells comprising afusion protein as described herein and an extended guide RNA. The cellsmay be transformed with the vectors comprising the fusion proteins,napDNAbps, reverse transcriptase, and extended guide RNAs. These geneticelements may be comprised on the same vectors or on different vectors.

In yet another aspect, the specification provides pharmaceuticalcompositions. In certain embodiments, the pharmaceutical compositionscomprise one or more of a napDNAbp, a fusion protein, a reversetranscriptase, and an extended guide RNA. In certain embodiments, thefusion protein described herein and a pharmaceutically acceptableexcipient. In other embodiments, the pharmaceutical compositionscomprise any extend guide RNA described herein and a pharmaceuticallyacceptable excipient. In still other embodiments, the pharmaceuticalcompositions comprise any extend guide RNA described herein incombination with any fusion protein described herein and apharmaceutically acceptable excipient. In yet other embodiments, thepharmaceutical compositions comprise any polynucleotide sequenceencoding one or more of a napDNAbp, a fusion protein, a reversetranscriptase, and an extended guide RNA. In still other embodiments,the various components disclosed herein may be separated into one ormore pharmaceutical compositions. For example, a first pharmaceuticalcomposition may comprise a fusion protein or a napDNAbp, a secondpharmaceutical compositions may comprise a reverse transcriptase, and athird pharmaceutical composition may comprise an extended guide RNA.

In still a further aspect, the present disclosure provides kits. In oneembodiment, the kit comprises one or more polynucleotides encoding oneor more components, including a fusion protein, a napDNAbp, a reversetranscriptase, and an extended guide RNA. The kits may also comprisevectors, cells, and isolated preparations of polypeptides, including anyfusion protein, napDNAbp, or reverse transcriptase disclosed herein.

In yet another aspect, the present disclosure provides for methods ofusing the disclosed compositions of matter.

In one embodiment, the methods relate to a method for installing adesired nucleotide change in a double-stranded DNA sequence. The methodfirst comprises contacting the double-stranded DNA sequence with acomplex comprising a fusion protein and an extended guide RNA, whereinthe fusion protein comprises a napDNAbp and a reverse transcriptase andwherein the extended guide RNA comprises a reverse transcriptiontemplate sequence comprising the desired nucleotide change. Next, themethod involves nicking the double-stranded DNA sequence on thenon-target strand, thereby generating a free single-strand DNA having a3′ end. The method then involves hybridizing the 3′ end of the freesingle-strand DNA to the reverse transcription template sequence,thereby priming the reverse transcriptase domain. The method theninvolves polymerizing a strand of DNA from the 3′ end, therebygenerating a single-strand DNA flap comprising the desired nucleotidechange. Then, the method involves replacing an endogenous DNA strandadjacent the cut site with the single-strand DNA flap, therebyinstalling the desired nucleotide change in the double-stranded DNAsequence.

In other embodiments, the disclosure provides for a method forintroducing one or more changes in the nucleotide sequence of a DNAmolecule at a target locus, comprising contacting the DNA molecule witha nucleic acid programmable DNA binding protein (napDNAbp) and a guideRNA which targets the napDNAbp to the target locus, wherein the guideRNA comprises a reverse transcriptase (RT) template sequence comprisingat least one desired nucleotide change. Next, the method involvesforming an exposed 3′ end in a DNA strand at the target locus and thenhybridizing the exposed 3′ end to the RT template sequence to primereverse transcription. Next, a single strand DNA flap comprising the atleast one desired nucleotide change based on the RT template sequence issynthesized or polymerized by reverse transcriptase. Lastly, the atleast one desired nucleotide change is incorporated into thecorresponding endogenous DNA, thereby introducing one or more changes inthe nucleotide sequence of the DNA molecule at the target locus.

In still other embodiments, the disclosure provides a method forintroducing one or more changes in the nucleotide sequence of a DNAmolecule at a target locus by target-primed reverse transcription, themethod comprising: (a) contacting the DNA molecule at the target locuswith a (i) fusion protein comprising a nucleic acid programmable DNAbinding protein (napDNAbp) and a reverse transcriptase and (ii) a guideRNA comprising an RT template comprising a desired nucleotide change;(b) conducting target-primed reverse transcription of the RT template togenerate a single strand DNA comprising the desired nucleotide change;and (c) incorporating the desired nucleotide change into the DNAmolecule at the target locus through a DNA repair and/or replicationprocess.

In certain embodiments, the step of replacing the endogenous DNA strandcomprises: (i) hybridizing the single-strand DNA flap to the endogenousDNA strand adjacent the cut site to create a sequence mismatch; (ii)excising the endogenous DNA strand; and (iii) repairing the mismatch toform the desired product comprising the desired nucleotide change inboth strands of DNA.

In various embodiments, the desired nucleotide change can be a singlenucleotide substitution (e.g., and transition or a transversion change),a deletion, or an insertion. For example, the desired nucleotide changecan be (1) a G to T substitution, (2) a G to A substitution, (3) a G toC substitution, (4) a T to G substitution, (5) a T to A substitution,(6) a T to C substitution, (7) a C to G substitution, (8) a C to Tsubstitution, (9) a C to A substitution, (10) an A to T substitution,(11) an A to G substitution, or (12) an A to C substitution.

In other embodiments, the desired nucleoid change can convert (1) a G:Cbasepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) aG:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5)a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair,(7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:Abasepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair toa T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:Tbasepair to a C:G basepair.

In still other embodiments, the method introduces a desired nucleotidechange that is an insertion. In certain cases, the insertion is at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least200, at least 300, at least 400, or at least 500 nucleotides in length.

In other embodiments, the method introduces a desired nucleotide changethat is a deletion. In certain other cases, the deletion is at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, at least 100, at least 200,at least 300, at least 400, or at least 500 nucleotides in length.

In various embodiments, the desired nucleotide change corrects adisease-associated gene. The disease-associated gene can be associatedwith a monogenetic disorder selected from the group consisting of:Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency;Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; MarfanSyndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease;Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease. In other embodiments,the disease-associated gene can be associated with a polygenic disorderselected from the group consisting of: heart disease; high bloodpressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

The methods disclosed herein may involve fusion proteins having anapDNAbp that is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9),or a nuclease active Cas9. In other embodiments, a napDNAbp and reversetranscriptase are not encoded as a single fusion protein, but rather canbe provided in separate constructs. Thus, in some embodiments, thereverse transcriptase can be provided in trans relative to the napDNAbp(rather than by way of a fusion protein).

In various embodiments involving methods, the napDNAbp may comprise anamino acid sequence of SEQ ID NOs: 26-61, 75-76, 126, 130, 137, 141,147, 153, 157, 445, 460, 467, and 482-487 (Cas9); (SpCas9); SEQ ID NO:77-86 (CP-Cas9); SEQ ID NO: 18-25 and 87-88 (SpCas9); and SEQ ID NOs:62-72 (Cas12). The napDNAbp may also comprise an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the aminoacid sequence of any one of SEQ ID NOs: 26-61, 75-76, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487 (Cas9); (SpCas9); SEQ IDNO: 77-86 (CP-Cas9); SEQ ID NO: 18-25 and 87-88 (SpCas9); and SEQ IDNOs: 62-72 (Cas12).

In various embodiments involving methods, the reverse transcriptase maycomprise any one of the amino acid sequences of SEQ ID NOs: 89-100,105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662,700-716, 739-742, and 766. The reverse transcriptase may also comprisean amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NOs: 89-100,105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662,700-716, 739-742, and 766.

The methods may involve the use of a PEgRNA comprising a nucleotidesequence of SEQ ID NOs: 101-104, 131, 181-183, 222-234, 237-244, 277,324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649,678-692, 735-736, 738, 757-761, 776-777, 2997-3103, 3113-3121,3305-3455, 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874,3890-3901, 3905-3911, 3913-3929, and 3972-3989, or a nucleotide sequencehaving at least 80%, or at least 85%, or at least 90%, or at least 95%,or at least 99% sequence identity thereto. The methods may comprise theuse of extended guide RNAs that comprise an RNA extension at the 3′ end,wherein the RNA extension comprises the reverse transcription templatesequence.

The methods may comprise the use of extended guide RNAs that comprise anRNA extension at the 5′ end, wherein the RNA extension comprises thereverse transcription template sequence.

The methods may comprise the use of extended guide RNAs that comprise anRNA extension at an intramolecular location in the guide RNA, whereinthe RNA extension comprises the reverse transcription template sequence.

The methods may comprise the use of extended guide RNAs having one ormore RNA extensions that are at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 200, at least 300, at least 400,or at least 500 nucleotides in length.

It should be appreciated that the foregoing concepts, and additionalconcepts discussed below, may be arranged in any suitable combination,as the present disclosure is not limited in this respect. Further, otheradvantages and novel features of the present disclosure will becomeapparent from the following detailed description of various non-limitingembodiments when considered in conjunction with the accompanyingfigures.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A provides a schematic of an exemplary process for introducing asingle nucleotide change, and/or insertion, and/or deletion into a DNAmolecule (e.g., a genome) using a fusion protein comprising a reversetranscriptase fused to a Cas9 protein in complex with an extended guideRNA molecule. In this embodiment, the guide RNA is extended at the 3′end to include a reverse transcriptase template sequence. The schematicshows how a reverse transcriptase (RT) fused to a Cas9 nickase, in acomplex with a guide RNA (gRNA), binds the DNA target site and nicks thePAM-containing DNA strand adjacent to the target nucleotide. The RTenzyme uses the nicked DNA as a primer for DNA synthesis from the gRNA,which is used as a template for the synthesis of a new DNA strand thatencodes the desired edit. The editing process shown may be referred toas target-primed reverse transcription editing (TRT editing) orequivalently, “prime editing.”

FIG. 1B provides the same representation as in FIG. 1A, except that theprime editor complex is represented more generally as[napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNA, wherein “P” refers toany polymerase (e.g., a reverse transcriptase), “napDNAbp” refers to anucleic acid programmable DNA binding protein (e.g., SpCas9), and“PEgRNA” refers to a prime editing guide RNA, and “]-[” refers to anoptional linker. As described elsewhere, e.g., FIGS. 3A-3G, the PEgRNAcomprises an 5′ extension arm comprising a primer binding site and a DNAsynthesis template. Although not shown, it is contemplated that theextension arm of the PEgRNA (i.e., which comprises a primer binding siteand a DNA synthesis template) can be DNA or RNA. The particularpolymerase contemplated in this configuration will depend upon thenature of the DNA synthesis template. For instance, if the DNA synthesistemplate is RNA, then the polymerase case be an RNA-dependent DNApolymerase (e.g., reverse transcriptase). If the DNA synthesis templateis DNA, then the polymerase can be a DNA-dependent DNA polymerase.

FIG. 1C provides a schematic of an exemplary process for introducing asingle nucleotide change, and/or insertion, and/or deletion into a DNAmolecule (e.g., a genome) using a fusion protein comprising a reversetranscriptase fused to a Cas9 protein in complex with an extended guideRNA molecule. In this embodiment, the guide RNA is extended at the 5′end to include a reverse transcriptase template sequence. The schematicshows how a reverse transcriptase (RT) fused to a Cas9 nickase, in acomplex with a guide RNA (gRNA), binds the DNA target site and nicks thePAM-containing DNA strand adjacent to the target nucleotide. The RTenzyme uses the nicked DNA as a primer for DNA synthesis from the gRNA,which is used as a template for the synthesis of a new DNA strand thatencodes the desired edit. The editing process shown may be referred toas target-primed reverse transcription editing (TRT editing) orequivalently, “prime editing.”

FIG. 1D provides the same representation as in FIG. 1C, except that theprime editor complex is represented more generally as[napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNA, wherein “P” refers toany polymerase (e.g., a reverse transcriptase), “napDNAbp” refers to anucleic acid programmable DNA binding protein (e.g., SpCas9), and“PEgRNA” refers to a prime editing guide RNA, and “]-[” refers to anoptional linker. As described elsewhere, e.g., FIGS. 3A-3G, the PEgRNAcomprises an 3′ extension arm comprising a primer binding site and a DNAsynthesis template. Although not shown, it is contemplated that theextension arm of the PEgRNA (i.e., which comprises a primer binding siteand a DNA synthesis template) can be DNA or RNA. The particularpolymerase contemplated in this configuration will depend upon thenature of the DNA synthesis template. For instance, if the DNA synthesistemplate is RNA, then the polymerase case be an RNA-dependent DNApolymerase (e.g., reverse transcriptase). If the DNA synthesis templateis DNA, then the polymerase can be a DNA-dependent DNA polymerase. Invarious embodiments, the PEgRNA can be engineered or synthesized toincorporate a DNA-based DNA synthesis template.

FIG. 1E is a schematic depicting an exemplary process of how thesynthesized single strand of DNA (which comprises the desired nucleotidechange) becomes resolved such that the desired nucleotide change isincorporated into the DNA. As shown, following synthesis of the editedstrand (or “mutagenic strand”), equilibration with the endogenousstrand, flap cleavage of the endogenous strand, and ligation leads toincorporation of the DNA edit after resolution of the mismatched DNAduplex through the action of endogenous DNA repair and/or replicationprocesses.

FIG. 1F is a schematic showing that “opposite strand nicking” can beincorporated into the resolution method of FIG. 1E to help drive theformation of the desired product versus the reversion product. Inopposite strand nicking, a second Cas9/gRNA complex is used to introducea second nick on the opposite strand from the initial nicked strand.This induces the endogenous cellular DNA repair and/or replicationprocesses to preferentially replace the unedited strand (i.e., thestrand containing the second nick site).

FIG. 1G provides another schematic of an exemplary process forintroducing a single nucleotide change, and/or insertion, and/ordeletion into a DNA molecule (e.g., a genome) of a target locus using anucleic acid programmable DNA binding protein (napDNAbp) complexed withan extended guide RNA. This process may be referred to as an embodimentof prime editing. The extended guide RNA comprises an extension at the3′ or 5′ end of the guide RNA, or at an intramolecular location in theguide RNA. In step (a), the napDNAbp/gRNA complex contacts the DNAmolecule and the gRNA guides the napDNAbp to bind to the target locus.In step (b), a nick in one of the strands of DNA (the R-loop strand, orthe PAM-containing strand, or the non-target DNA strand, or theprotospacer strand) of the target locus is introduced (e.g., by anuclease or chemical agent), thereby creating an available 3′ end in oneof the strands of the target locus. In certain embodiments, the nick iscreated in the strand of DNA that corresponds to the R-loop strand,i.e., the strand that is not hybridized to the guide RNA sequence. Instep (c), the 3′ end DNA strand interacts with the extended portion ofthe guide RNA in order to prime reverse transcription. In certainembodiments, the 3′ ended DNA strand hybridizes to a specific RT primingsequence on the extended portion of the guide RNA. In step (d), areverse transcriptase is introduced which synthesizes a single strand ofDNA from the 3′ end of the primed site towards the 3′ end of the guideRNA. This forms a single-strand DNA flap comprising the desirednucleotide change (e.g., the single base change, insertion, or deletion,or a combination thereof). In step (e), the napDNAbp and guide RNA arereleased. Steps (f) and (g) relate to the resolution of the singlestrand DNA flap such that the desired nucleotide change becomesincorporated into the target locus. This process can be driven towardsthe desired product formation by removing the corresponding 5′endogenous DNA flap that forms once the 3′ single strand DNA flapinvades and hybridizes to the complementary sequence on the otherstrand. The process can also be driven towards product formation withsecond strand nicking, as exemplified in FIG. 1F. This process mayintroduce at least one or more of the following genetic changes:transversions, transitions, deletions, and insertions.

FIG. 1H is a schematic depicting the types of genetic changes that arepossible with the prime editing processes described herein. The types ofnucleotide changes achievable by prime editing include deletions(including short and long deletions), single-nucleotide changes(including transitions and transversions), inversions, and insertions(including short and long deletions).

FIG. 1I is a schematic depicting temporal second strand nickingexemplified by PE3b (PE3b=PE2 prime editor fusion protein+PEgRNA+secondstrand nicking guide RNA). Temporal second strand nicking is a variantof second strand nicking in order to facilitate the formation of thedesired edited product. The “temporal” term refers to the fact that thesecond-strand nick to the unedited strand occurs only after the desirededit is installed in the edited strand. This avoids concurrent nicks onboth strands to lead to double-stranded DNA breaks.

FIGS. 1J-1K depict a variation of prime editing contemplated herein thatreplaces the napDNAbp (e.g., SpCas9 nickase) with any programmablenuclease domain, such as zinc finger nucleases (ZFN) or transcriptionactivator-like effector nucleases (TALEN). As such, it is contemplatedthat suitable nucleases do not necessarily need to be “programmed” by anucleic acid targeting molecule (such as a guide RNA), but rather, maybe programmed by defining the specificity of a DNA-binding domain, suchas and in particular, a nuclease. Just as in prime editing with napDNAbpmoieties, it is preferable that such alternative programmable nucleasesbe modified such that only one strand of a target DNA is cut. In otherwords, the programmable nucleases should function as nickases,preferably. Once a programmable nuclease is selected (e.g., a ZFN or aTALEN), then additional functionalities may be engineered into thesystem to allow it to operate in accordance with a prime editing-likemechanism. For example, the programmable nucleases may be modified bycoupling (e.g., via a chemical linker) an RNA or DNA extension armthereto, wherein the extension arm comprises a primer binding site (PBS)and a DNA synthesis template. The programmable nuclease may also becoupled (e.g., via a chemical or amino acid linker) to a polymerase, thenature of which will depend upon whether the extension arm is DNA orRNA. In the case of an RNA extension arm, the polymerase can be anRNA-dependent DNA polymerase (e.g., reverse transcriptase). In the caseof a DNA extension arm, the polymerase can be a DNA-dependent DNApolymerase (e.g., a prokaryotic polymerase, including Pol I, Pol II, orPol III, or a eukaryotic polymerase, including Pol a, Pol b, Pol g, Pold, Pol e, or Pol z). The system may also include other functionalitiesadded as fusions to the programmable nucleases, or added in trans tofacilitate the reaction as a whole (e.g., (a) a helicase to unwind theDNA at the cut site to make the cut strand with the 3′ end available asa primer, (b) a flap endonuclease (e.g., FEN1) to help remove theendogenous strand on the cut strand to drive the reaction towardsreplacement of the endogenous strand with the synthesized strand, or (c)a nCas9:gRNA complex to create a second site nick on the oppositestrand, which may help drive the integration of the synthesize repairthrough favored cellular repair of the non-edited strand). In ananalogous manner to prime editing with a napDNAbp, such a complex withan otherwise programmable nuclease could be used to synthesize and theninstall a newly synthesized replacement strand of DNA carrying an editof interest permanently into a target site of DNA.

FIG. 1L depicts, in one embodiment, the anatomical features of a targetDNA that may be edited by prime editing. The target DNA comprises a“non-target strand” and a “target strand.” The target-strand is thestrand that becomes annealed to the spacer of a PEgRNA of a prime editorcomplex that recognizes the PAM site (in this case, NGG, which isrecognized by the canonical SpCas9-based prime editors) The targetstrand may also be referred to as the “non-PAM strand” or the “non-editstrand.” By contrast, the non-target strand (i.e., the strand containingthe protospacer and the PAM sequence of NGG) may be referred to as the“PAM-strand” or the “edit strand.” In various embodiments, the nick siteof the PE complex will be in the protospacer on the PAM-strand (e.g.,with the SpCas9-based PE). The location of the nick will becharacteristic of the particular Cas9 that forms the PE. For example,with an SpCas9-based PE, the nick site in the phosphodiester bondbetween bases three (“−3” position relative to the position 1 of the PAMsequence) and four (“−4” position relative to position 1 of the PAMsequence). The nick site in the protospacer forms a free 3′ hydroxylgroup, which as seen in the following figures, complexes with the primerbinding site of the extension arm of the PEgRNA and provides thesubstrate to begin polymerization of a single strand of DNA code for bythe DNA synthesis template of the extension arm of the PEgRNA. Thispolymerization reaction is catalyzed by the polymerase (e.g., reversetranscriptase) of the PE fusion protein in the 5′ to 3′ direction.Polymerization terminates before reaching the gRNA core (e.g., byinclusion of a polymerization termination signal, or secondarystructure, which functions to terminate the polymerization activity ofPE), producing a single strand DNA flap that is extended from theoriginal 3′ hydroxyl group of the nicked PAM strand. The DNA synthesistemplate codes for a single strand DNA that is homologous to theendogenous 5′-ended single strand of DNA that immediately follows thenick site on the PAM strand and incorporates the desired nucleotidechange (e.g., single base substitution, insertion, deletion, inversion).The position of the desired edit can be in any position followingdownstream of the nick site on the PAM strand, which can includeposition +1, +2, +3, +4 (the start of the PAM site), +5 (position 2 ofthe PAM site), +6 (position 3 of the PAM site), +7, +8, +9, +10, +11,+12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25,+26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39,+40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50, +51, +52, +53,+54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67,+68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81,+82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95,+96, +97, +98, +99, +100, +101, +102, +103, +104, +105, +106, +107,+108, +109, +110, +111, +112, +113, +114, +115, +116, +117, +118, +119,+120, +121, +122, +123, +124, +125, +126, +127, +128, +129, +130, +131,+132, +133, +134, +135, +136, +137, +138, +139, +140, +141, +142, +143,+144, +145, +146, +147, +148, +149, or +150, or more (relative to thedownstream position of the nick site). Once the 3′end single strandedDNA (containing the edit of interest) replaces the endogenous 5′ endsingle stranded DNA, the DNA repair and replication processes willresult in permanent installation of the edit site on the PAM strand, andthen correction of the mismatch on the non-PAM strand that will exist atthe edit site. In this way, the edit will extend to both strands of DNAon the target DNA site. It will be appreciated that reference to “editedstrand” and “non-edited” strand only intends to delineate the strands ofDNA involved in the PE mechanism. The “edited strand” is the strand thatfirst becomes edited by replacement of the 5′ ended single strand DNAimmediately downstream of the nick site with the synthesized 3′ endedsingle stranded DNA containing the desired edit. The “non-edited” strandis the strand pair with the edited strand, but which itself also becomesedited through repair and/or replication to be complementary to theedited strand, and in particular, the edit of interest.

FIG. 1M depicts the mechanism of prime editing showing the anatomicalfeatures of the target DNA, prime editor complex, and the interactionbetween the PEgRNA and the target DNA. First, a prime editor comprisinga fusion protein having a polymerase (e.g., reverse transcriptase) and anapDNAbp (e.g., SpCas9 nickase, e.g., a SpCas9 having a deactivatingmutation in an HNH nuclease domain (e.g., H840A) or a deactivatingmutation in a RuvC nuclease domain (D10A)) is complexed with a PEgRNAand DNA having a target DNA to be edited. The PEgRNA comprises a spacer,gRNA core (aka gRNA scaffold or gRNA backbone) (which binds to thenapDNAbp), and an extension arm. The extension arm can be at the 3′ end,the 5′ end, or somewhere within the PEgRNA molecule. As shown, theextension arm is at the 3′ end of the PEgRNA. The extension armcomprises in the 3′ to 5′ direction a primer binding site and a DNAsynthesis template (comprising both an edit of interest and regions ofhomology (i.e., homology arms) that are homologous with the 5′ endedsingle stranded DNA immediately following the nick site on the PAMstrand. As shown, once the nick is introduced thereby producing a free3′ hydroxyl group immediately upstream of the nick site, the regionimmediately upstream of the nick site on the PAM strand anneals to acomplementary sequence at the 3′ end of the extension arm referred to asthe “primer binding site,” creating a short double-stranded region withan available 3′ hydroxyl end, which forms a substrate for the polymeraseof the prime editor complex. The polymerase (e.g., reversetranscriptase) then polymerase as strand of DNA from the 3′ hydroxyl endto the end of the extension arm. The sequence of the single stranded DNAis coded for by the DNA synthesis template, which is the portion of theextension arm (i.e., excluding the primer binding site) that is “read”by the polymerase to synthesize new DNA. This polymerization effectivelyextends the sequence of the original 3′ hydroxyl end of the initial nicksite. The DNA synthesis template encodes a single strand of DNA thatcomprises not only the desired edit, but also regions that arehomologous to the endogenous single strand of DNA immediately downstreamof the nick site on the PAM strand. Next, the encoded 3′ ended singlestrand of DNA (i.e., the 3′ single strand DNA flap) displaces thecorresponding homologous endogenous 5′-ended single strand of DNAimmediately downstream of the nick site on the PAM strand, forming a DNAintermediate having a 5′-ended single strand DNA flap, which is removedby the cell (e.g., by a flap endonuclease). The 3′-ended single strandDNA flap, which anneals to the complement of the endogenous 5′-endedsingle strand DNA flap, is ligated to the endogenous strand after the 5′DNA flap is removed. The desired edit in the 3′ ended single strand DNAflap, now annealed and ligate, forms a mismatch with the complementstrand, which undergoes DNA repair and/or a round of replication,thereby permanently installing the desired edit on both strands.

FIG. 2 shows three Cas complexes (SpCas9, SaCas9, and LbCas12a) that canbe used in the herein described prime editors and their PAM, gRNA, andDNA cleavage features. The figure shows designs for complexes involvingSpCas9, SaCas9, and LbCas12a.

FIGS. 3A-3F show designs for engineered 5′ prime editor gRNA (FIG. 3A),3′ prime editor gRNA (FIG. 3B), and an intramolecular extension (FIG.3C). The extended guide RNA (or extended gRNA) may also be referred toherein as PEgRNA or “prime editing guide RNA.” FIG. 3D and FIG. 3Eprovide additional embodiments of 3′ and 5′ prime editor gRNAs(PEgRNAs), respectively. FIG. 3F illustrates the interaction between a3′ end prime editor guide RNA with a target DNA sequence. Theembodiments of FIGS. 3A-3C depict exemplary arrangements of the reversetranscription template sequence (i.e., or more broadly referred to as aDNA synthesis template, as indicated, since the RT is only one type ofpolymerase that may be used in the context of prime editors), the primerbinding site, and an optional linker sequence in the extended portionsof the 3′, 5′, and intramolecular versions, as well as the generalarrangements of the spacer and core regions. The disclosed prime editingprocess is not limited to these configurations of extended guide RNAs.The embodiment of FIG. 3D provides the structure of an exemplary PEgRNAcontemplated herein. The PEgRNA comprises three main component elementsordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and anextension arm at the 3′ end. The extension arm may further be dividedinto the following structural elements in the 5′ to 3′ direction,namely: a primer binding site (A), an edit template (B), and a homologyarm (C). In addition, the PEgRNA may comprise an optional 3′ endmodifier region (e1) and an optional 5′ end modifier region (e2). Stillfurther, the PEgRNA may comprise a transcriptional termination signal atthe 3′ end of the PEgRNA (not depicted). These structural elements arefurther defined herein. The depiction of the structure of the PEgRNA isnot meant to be limiting and embraces variations in the arrangement ofthe elements. For example, the optional sequence modifiers (e1) and (e2)could be positioned within or between any of the other regions shown,and not limited to being located at the 3′ and 5′ ends. The PEgRNA couldcomprise, in certain embodiments, secondary RNA structure, such as, butnot limited to, hairpins, stem/loops, toe loops, RNA-binding proteinrecruitment domains (e.g., the MS2 aptamer which recruits and binds tothe MS2cp protein). For instance, such secondary structures could beposition within the spacer, the gRNA core, or the extension arm, and inparticular, within the e1 and/or e2 modifier regions. In addition tosecondary RNA structures, the PEgRNAs could comprise (e.g., within thee1 and/or e2 modifier regions) a chemical linker or a poly(N) linker ortail, where “N” can be any nucleobase. In some embodiments (e.g., asshown in FIG. 72(c)), the chemical linker may function to preventreverse transcription of the sgRNA scaffold or core. In addition, incertain embodiments (e.g., see FIG. 72(c)), the extension arm (3) couldbe comprised of RNA or DNA, and/or could include one or more nucleobaseanalogs (e.g., which might add functionality, such as temperatureresilience). Still further, the orientation of the extension arm (3) canbe in the natural 5′-to-3′ direction, or synthesized in the oppositeorientation in the 3′-to-5′ direction (relative to the orientation ofthe PEgRNA molecule overall). It is also noted that one of ordinaryskill in the art will be able to select an appropriate DNA polymerase,depending on the nature of the nucleic acid materials of the extensionarm (i.e., DNA or RNA), for use in prime editing that may be implementedeither as a fusion with the napDNAbp or as provided in trans as aseparate moiety to synthesize the desired template-encoded 3′single-strand DNA flap that includes the desired edit. For example, ifthe extension arm is RNA, then the DNA polymerase could be a reversetranscriptase or any other suitable RNA-dependent DNA polymerase.However, if the extension arm is DNA, then the DNA polymerase could be aDNA-dependent DNA polymerase. In various embodiments, provision of theDNA polymerase could be in trans, e.g., through the use of anRNA-protein recruitment domain (e.g., an MS2 hairpin installed on thePEgRNA (e.g., in the e1 or e2 region, or elsewhere and an MS2cp proteinfused to the DNA polymerase, thereby co-localizing the DNA polymerase tothe PEgRNA). It is also noted that the primer binding site does notgenerally form a part of the template that is used by the DNA polymerase(e.g., reverse transcriptase) to encode the resulting 3′ single-strandDNA flap that includes the desired edit. Thus, the designation of the“DNA synthesis template” refers to the region or portion of theextension arm (3) that is used as a template by the DNA polymerase toencode the desired 3′ single-strand DNA flap containing the edit andregions of homology to the 5′ endogenous single strand DNA flap that isreplaced by the 3′ single strand DNA strand product of prime editing DNAsynthesis. In some embodiments, the DNA synthesis template includes the“edit template” and the “homology arm”, or one or more homology arms,e.g., before and after the edit template. The edit template can be assmall as a single nucleotide substitution, or it may be an insertion, oran inversion of DNA. In addition, the edit template may also include adeletion, which can be engineered by encoding homology arm that containsa desired deletion. In other embodiments, the DNA synthesis template mayalso include the e2 region or a portion thereof. For instance, if the e2region comprises a secondary structure that causes termination of DNApolymerase activity, then it is possible that DNA polymerase functionwill be terminated before any portion of the e2 region is actual encodedinto DNA. It is also possible that some or even all of the e2 regionwill be encoded into DNA. How much of e2 is actually used as a templatewill depend on its constitution and whether that constitution interruptsDNA polymerase function.

The embodiment of FIG. 3E provides the structure of another PEgRNAcontemplated herein. The PEgRNA comprises three main component elementsordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and anextension arm at the 3′ end. The extension arm may further be dividedinto the following structural elements in the 5′ to 3′ direction,namely: a primer binding site (A), an edit template (B), and a homologyarm (C). In addition, the PEgRNA may comprise an optional 3′ endmodifier region (e1) and an optional 5′ end modifier region (e2). Stillfurther, the PEgRNA may comprise a transcriptional termination signal onthe 3′ end of the PEgRNA (not depicted). These structural elements arefurther defined herein. The depiction of the structure of the PEgRNA isnot meant to be limiting and embraces variations in the arrangement ofthe elements. For example, the optional sequence modifiers (e1) and (e2)could be positioned within or between any of the other regions shown,and not limited to being located at the 3′ and 5′ ends. The PEgRNA couldcomprise, in certain embodiments, secondary RNA structures, such as, butnot limited to, hairpins, stem/loops, toe loops, RNA-binding proteinrecruitment domains (e.g., the MS2 aptamer which recruits and binds tothe MS2cp protein). These secondary structures could be positionedanywhere in the PEgRNA molecule. For instance, such secondary structurescould be position within the spacer, the gRNA core, or the extensionarm, and in particular, within the e1 and/or e2 modifier regions. Inaddition to secondary RNA structures, the PEgRNAs could comprise (e.g.,within the e1 and/or e2 modifier regions) a chemical linker or a poly(N)linker or tail, where “N” can be any nucleobase. In some embodiments(e.g., as shown in FIG. 72(c)), the chemical linker may function toprevent reverse transcription of the sgRNA scaffold or core. Inaddition, in certain embodiments (e.g., see FIG. 72(c)), the extensionarm (3) could be comprised of RNA or DNA, and/or could include one ormore nucleobase analogs (e.g., which might add functionality, such astemperature resilience). Still further, the orientation of the extensionarm (3) can be in the natural 5′-to-3′ direction, or synthesized in theopposite orientation in the 3′-to-5′ direction (relative to theorientation of the PEgRNA molecule overall). It is also noted that oneof ordinary skill in the art will be able to select an appropriate DNApolymerase, depending on the nature of the nucleic acid materials of theextension arm (i.e., DNA or RNA), for use in prime editing that may beimplemented either as a fusion with the napDNAbp or as provided in transas a separate moiety to synthesize the desired template-encoded 3′single-strand DNA flap that includes the desired edit. For example, ifthe extension arm is RNA, then the DNA polymerase could be a reversetranscriptase or any other suitable RNA-dependent DNA polymerase.However, if the extension arm is DNA, then the DNA polymerase could be aDNA-dependent DNA polymerase. In various embodiments, provision of theDNA polymerase could be in trans, e.g., through the use of anRNA-protein recruitment domain (e.g., an MS2 hairpin installed on thePEgRNA (e.g., in the e1 or e2 region, or elsewhere and an MS2cp proteinfused to the DNA polymerase, thereby co-localizing the DNA polymerase tothe PEgRNA). It is also noted that the primer binding site does notgenerally form a part of the template that is used by the DNA polymerase(e.g., reverse transcriptase) to encode the resulting 3′ single-strandDNA flap that includes the desired edit. Thus, the designation of the“DNA synthesis template” refers to the region or portion of theextension arm (3) that is used as a template by the DNA polymerase toencode the desired 3′ single-strand DNA flap containing the edit andregions of homology to the 5′ endogenous single strand DNA flap that isreplaced by the 3′ single strand DNA strand product of prime editing DNAsynthesis. In some embodiments, the DNA synthesis template includes the“edit template” and the “homology arm”, or one or more homology arms,e.g., before and after the edit template. The edit template can be assmall as a single nucleotide substitution, or it may be an insertion, oran inversion of DNA. In addition, the edit template may also include adeletion, which can be engineered by encoding homology arm that containsa desired deletion. In other embodiments, the DNA synthesis template mayalso include the e2 region or a portion thereof. For instance, if the e2region comprises a secondary structure that causes termination of DNApolymerase activity, then it is possible that DNA polymerase functionwill be terminated before any portion of the e2 region is actual encodedinto DNA. It is also possible that some or even all of the e2 regionwill be encoded into DNA. How much of e2 is actually used as a templatewill depend on its constitution and whether that constitution interruptsDNA polymerase function.

The schematic of FIG. 3F depicts the interaction of a typical PEgRNAwith a target site of a double stranded DNA and the concomitantproduction of a 3′ single stranded DNA flap containing the geneticchange of interest. The double strand DNA is shown with the top strand(i.e., the target strand) in the 3′ to 5′ orientation and the lowerstrand (i.e., the PAM strand or non-target strand) in the 5′ to 3′direction. The top strand comprises the complement of the “protospacer”and the complement of the PAM sequence and is referred to as the “targetstrand” because it is the strand that is target by and anneals to thespacer of the PEgRNA. The complementary lower strand is referred to asthe “non-target strand” or the “PAM strand” or the “protospacer strand”since it contains the PAM sequence (e.g., NGG) and the protospacer.Although not shown, the PEgRNA depicted would be complexed with a Cas9or equivalent domain of a prime editor fusion protein. As shown in theschematic, the spacer of the PEgRNA anneals to the complementary regionof the protospacer on the target strand. This interaction forms asDNA/RNA hybrid between the spacer RNA and the complement of theprotospacer DNA, and induces the formation of an R loop in theprotospacer. As taught elsewhere herein, the Cas9 protein (not shown)then induces a nick in the non-target strand, as shown. This then leadsto the formation of the 3′ ssDNA flap region immediately upstream of thenick site which, in accordance with *z*, interacts with the 3′ end ofthe PEgRNA at the primer binding site. The 3′ end of the ssDNA flap(i.e., the reverse transcriptase primer sequence) anneals to the primerbinding site (A) on the PEgRNA, thereby priming reverse transcriptase.Next, reverse transcriptase (e.g., provided in trans or provided cis asa fusion protein, attached to the Cas9 construct) then polymerizes asingle strand of DNA which is coded for by the DNA synthesis template(including the edit template (B) and homology arm (C)). Thepolymerization continues towards the 5′ end of the extension arm. Thepolymerized strand of ssDNA forms a ssDNA 3′ end flap which, as describeelsewhere (e.g., as shown in FIG. 1G), invades the endogenous DNA,displacing the corresponding endogenous strand (which is removed as a 5′ended DNA flap of endogenous DNA), and installing the desired nucleotideedit (single nucleotide base pair change, deletions, insertions(including whole genes) through naturally occurring DNArepair/replication rounds.

FIG. 3G depicts yet another embodiment of prime editing contemplatedherein. In particular, the top schematic depicts one embodiment of aprime editor (PE), which comprises a fusion protein of a napDNAbp (e.g.,SpCas9) and a polymerase (e.g., a reverse transcriptase), which arejoined by a linker. The PE forms a complex with a PEgRNA by binding tothe gRNA core of the PEgRNA. In the embodiment shown, the PEgRNA isequipped with a 3′ extension arm that comprises, beginning at the 3′end, a primer binding site (PBS) followed by a DNA synthesis template.The bottom schematic depicts a variant of a prime editor, referred to asa “trans prime editor (tPE).” In this embodiment, the DNA synthesistemplate and PBS are decoupled from the PEgRNA and presented on aseparate molecule, referred to as a trans prime editor RNA template(“tPERT”), which comprises an RNA-protein recruitment domain (e.g., aMS2 hairpin). The PE itself is further modified to comprise a fusion toa rPERT recruiting protein (“RP”), which is a protein which specificallyrecognizes and binds to the RNA-protein recruitment domain. In theexample where the RNA-protein recruitment domain is an MS2 hairpin, thecorresponding rPERT recruiting protein can be MS2cp of the MS2 taggingsystem. The MS2 tagging system is based on the natural interaction ofthe MS2 bacteriophage coat protein (“MCP” or “MS2cp”) with a stem-loopor hairpin structure present in the genome of the phage, i.e., the “MS2hairpin” or “MS2 aptamer.” In the case of trans prime editing, theRP-PE:gRNA complex “recruits” a tPERT having the appropriate RNA-proteinrecruitment domain to co-localize with the PE:gRNA complex, therebyproviding the PBS and DNA synthesis template in trans for use in primeediting, as shown in the example depicted in FIG. 3H.

FIG. 3H depicts the process of trans prime editing. In this embodiment,the trans prime editor comprises a “PE2” prime editor (i.e., a fusion ofa Cas9(H840A) and a variant MMLV RT) fused to an MS2cp protein (i.e., atype of recruiting protein that recognizes and binds to an MS2 aptamer)and which is complexed with an sgRNA (i.e., a standard guide RNA asopposed to a PEgRNA). The trans prime editor binds to the target DNA andnicks the nontarget strand. The MS2cp protein recruits a tPERT in transthrough the specific interaction with the RNA-protein recruitment domainon the tPERT molecule. The tPERT becomes co-localized with the transprime editor, thereby providing the PBS and DNA synthesis templatefunctions in trans for use by the reverse transcriptase polymerase tosynthesize a single strand DNA flap having a 3′ end and containing thedesired genetic information encoded by the DNA synthesis template.

FIGS. 4A-4E demonstrate in vitro TPRT assays (i.e., prime editingassays). FIG. 4A is a schematic of fluorescently labeled DNA substratesgRNA templated extension by an RT enzyme, PAGE. FIG. 4B shows TPRT(i.e., prime editing) with pre-nicked substrates, dCas9, and 5′-extendedgRNAs of differing synthesis template length. FIG. 4C shows the RTreaction with pre-nicked DNA substrates in the absence of Cas9. FIG. 4Dshows TPRT (i.e., prime editing) on full dsDNA substrates withCas9(H840A) and 5′-extended gRNAs. FIG. 4E shows a 3′-extended gRNAtemplate with pre-nicked and full dsDNA substrates. All reactions arewith M-MLV RT.

FIG. 5 shows in vitro validations using 5′-extended gRNAs with varyinglength synthesis templates. Fluorescently labeled (Cy5) DNA targets wereused as substrates, and were pre-nicked in this set of experiments. TheCas9 used in these experiments is catalytically dead Cas9 (dCas9), andthe RT used is Superscript III, a commercial RT derived from theMoloney-Murine Leukemia Virus (M-MLV). dCas9:gRNA complexes were formedfrom purified components. Then, the fluorescently labeled DNA substratewas added along with dNTPs and the RT enzyme. After 1 hour of incubationat 37° C., the reaction products were analyzed by denaturingurea-polyacrylamide gel electrophoresis (PAGE). The gel image showsextension of the original DNA strand to lengths that are consistent withthe length of the reverse transcription template.

FIG. 6 shows in vitro validations using 5′-extended gRNAs with varyinglength synthesis templates, which closely parallels those shown in FIG.5 . However, the DNA substrates are not pre-nicked in this set ofexperiments. The Cas9 used in these experiments is a Cas9 nickase(SpyCas9 H840A mutant) and the RT used is Superscript III, a commercialRT derived from the Moloney-Murine Leukemia Virus (M-MLV). The reactionproducts were analyzed by denaturing urea-polyacrylamide gelelectrophoresis (PAGE). As shown in the gel, the nickase efficientlycleaves the DNA strand when the standard gRNA is used (gRNA_0, lane 3).

FIG. 7 demonstrates that 3′ extensions support DNA synthesis and do notsignificantly effect Cas9 nickase activity. Pre-nicked substrates (blackarrow) are near-quantitatively converted to RT products when eitherdCas9 or Cas9 nickase is used (lanes 4 and 5). Greater than 50%conversion to the RT product (white arrow) is observed with fullsubstrates (lane 3). Cas9 nickase (SpyCas9 H840A mutant), catalyticallydead Cas9 (dCas9) and Superscript III, a commercial RT derived from theMoloney-Murine Leukemia Virus (M-MLV) are used.

FIG. 8 demonstrates dual color experiments that were used to determineif the RT reaction preferentially occurs with the gRNA in cis (bound inthe same complex). Two separate experiments were conducted for5′-extended and 3′-extended gRNAs. Products were analyzed by PAGE.Product ratio calculated as (Cy3cis/Cy3trans)/(Cy5trans/Cy5cis).

FIGS. 9A-9D demonstrates a flap model substrate. FIG. 9A shows a dual-FPreporter for flap-directed mutagenesis. FIG. 9B shows stop codon repairin HEK cells. FIG. 9C shows sequenced yeast clones after flap repair.FIG. 9D shows testing of different flap features in human cells.

FIG. 10 demonstrates prime editing on plasmid substrates. Adual-fluorescent reporter plasmid was constructed for yeast (S.cerevisiae) expression. Expression of this construct in yeast producesonly GFP. The in vitro prime editing reaction introduces a pointmutation, and transforms the parent plasmid or an in vitro Cas9(H840A)nicked plasmid into yeast. The colonies are visualized by fluorescenceimaging. Yeast dual-FP plasmid transformants are shown. Transforming theparent plasmid or an in vitro Cas9(H840A) nicked plasmid results in onlyGFP expressing colonies. The prime editing reaction with 5′-extended or3′-extended gRNAs produces a mix of colonies. The latter express bothGFP and mCherry. More colonies are observed with the 3′-extended gRNA. Apositive control that contains no stop codon is shown as well.

FIG. 11 shows prime editing on plasmid substrates similar to theexperiment in FIG. 10 , but instead of installing a point mutation inthe stop codon, prime editing installs a single nucleotide insertion(left) or deletion (right) that repairs a frameshift mutation and allowsfor synthesis of downstream mCherry. Both experiments used 3′ extendedgRNAs.

FIG. 12 shows editing products of prime editing on plasmid substrates,characterized by Sanger sequencing. Individually colonies from the TRTtransformations were selected and analyzed by Sanger sequencing. Preciseedits were observed by sequencing select colonies. Colonies containedplasmids with the original DNA sequence, while colonies contained theprecise mutation designed by the prime editing gRNA. No other pointmutations or indels were observed.

FIG. 13 shows the potential scope for the new prime editing technologyis shown and compared to deaminase-mediated base editor technologies.

FIG. 14 shows a schematic of editing in human cells.

FIG. 15 demonstrates the extension of the primer binding site in gRNA.

FIG. 16 shows truncated gRNAs for adjacent targeting.

FIGS. 17A-17C are graphs displaying the % T to A conversion at thetarget nucleotide after transfection of components in human embryonickidney (HEK) cells. FIG. 17A shows data, which presents results using anN-terminal fusion of wild type MLV reverse transcriptase to Cas9(H840A)nickase (32-amino acid linker). FIG. 17B is similar to FIG. 17A, but forC-terminal fusion of the RT enzyme. FIG. 17C is similar to FIG. 17A butthe linker between the MLV RT and Cas9 is 60 amino acids long instead of32 amino acids.

FIG. 18 shows high purity T to A editing at HEK3 site by high-throughputamplicon sequencing. The output of sequencing analysis displays the mostabundant genotypes of edited cells.

FIG. 19 shows editing efficiency at the target nucleotide (striped bars)alongside indel rates (white bars). WT refers to the wild type MLV RTenzyme. The mutant enzymes (M1 through M4) contain the mutations listedto the right. Editing rates were quantified by high throughputsequencing of genomic DNA amplicons.

FIG. 20 shows editing efficiency of the target nucleotide when a singlestrand nick is introduced in the complementary DNA strand in proximityto the target nucleotide. Nicking at various distances from the targetnucleotide was tested (triangles). Editing efficiency at the target basepair (striped bars) is shown alongside the indel formation rate (whitebars). The “none” example does not contain a complementary strandnicking guide RNA. Editing rates were quantified by high throughputsequencing of genomic DNA amplicons.

FIG. 21 demonstrates processed high throughput sequencing data showingthe desired T to A transversion mutation and general absence of othermajor genome editing byproducts.

FIG. 22 provides a schematic of an exemplary process for conductingtargeted mutagenesis with an error-prone reverse transcriptase on atarget locus using a nucleic acid programmable DNA binding protein(napDNAbp) complexed with an extended guide RNA, i.e., prime editingwith an error-prone RT. This process may be referred to as an embodimentof prime editing for targeted mutagenesis. The extended guide RNAcomprises an extension at the 3′ or 5′ end of the guide RNA, or at anintramolecular location in the guide RNA. In step (a), the napDNAbp/gRNAcomplex contacts the DNA molecule and the gRNA guides the napDNAbp tobind to the target locus to be mutagenized. In step (b), a nick in oneof the strands of DNA of the target locus is introduced (e.g., by anuclease or chemical agent), thereby creating an available 3′ end in oneof the strands of the target locus. In certain embodiments, the nick iscreated in the strand of DNA that corresponds to the R-loop strand,i.e., the strand that is not hybridized to the guide RNA sequence. Instep (c), the 3′ end DNA strand interacts with the extended portion ofthe guide RNA in order to prime reverse transcription. In certainembodiments, the 3′ ended DNA strand hybridizes to a specific RT primingsequence on the extended portion of the guide RNA. In step (d), anerror-prone reverse transcriptase is introduced which synthesizes amutagenized single strand of DNA from the 3′ end of the primed sitetowards the 3′ end of the guide RNA. Exemplary mutations are indicatedwith an asterisk “*”. This forms a single-strand DNA flap comprising thedesired mutagenized region. In step (e), the napDNAbp and guide RNA arereleased. Steps (f) and (g) relate to the resolution of the singlestrand DNA flap (comprising the mutagenized region) such that thedesired mutagenized region becomes incorporated into the target locus.This process can be driven towards the desired product formation byremoving the corresponding 5′ endogenous DNA flap that forms once the 3′single strand DNA flap invades and hybridizes to the complementarysequence on the other strand. The process can also be driven towardsproduct formation with second strand nicking, as exemplified in FIG. 1F.Following endogenous DNA repair and/or replication processes, themutagenized region becomes incorporated into both strands of DNA of theDNA locus.

FIG. 23 is a schematic of gRNA design for contracting trinucleotiderepeat sequences and trinucleotide repeat contraction with TPRT genomeediting (i.e., prime editing). Trinucleotide repeat expansion isassociated with a number of human diseases, including Huntington'sdisease, Fragile X syndrome, and Friedreich's ataxia. The most commontrinucleotide repeat contains CAG triplets, though GAA triplets(Friedreich's ataxia) and CGG triplets (Fragile X syndrome) also occur.Inheriting a predisposition to expansion, or acquiring an alreadyexpanded parental allele, increases the likelihood of acquiring thedisease. Pathogenic expansions of trinucleotide repeats couldhypothetically be corrected using prime editing. A region upstream ofthe repeat region can be nicked by an RNA-guided nuclease, then used toprime synthesis of a new DNA strand that contains a healthy number ofrepeats (which depends on the particular gene and disease). After therepeat sequence, a short stretch of homology is added that matches theidentity of the sequence adjacent to the other end of the repeat (boldstrand). Invasion of the newly synthesized strand, and subsequentreplacement of the endogenous DNA with the newly synthesized flap, leadsto a contracted repeat allele.

FIG. 24 is a schematic showing precise 10-nucleotide deletion with primeediting. A guide RNA targeting the HEK3 locus was designed with areverse transcription template that encodes a 10-nucleotide deletionafter the nick site. Editing efficiency in transfected HEK cells wasassessed using amplicon sequencing.

FIG. 25 is a schematic showing gRNA design for peptide tagging genes atendogenous genomic loci and peptide tagging with TPRT genome editing(i.e., prime editing). The FlAsH and ReAsH tagging systems comprise twoparts: (1) a fluorophore-biarsenical probe, and (2) a geneticallyencoded peptide containing a tetracysteine motif, exemplified by thesequence FLNCCPGCCMEP (SEQ ID NO: 1). When expressed within cells,proteins containing the tetracysteine motif can be fluorescently labeledwith fluorophore-arsenic probes (see ref: J. Am. Chem. Soc., 2002, 124(21), pp 6063-6076. DOI: 10.1021/ja017687n). The “sortagging” systememploys bacterial sortase enzymes that covalently conjugate labeledpeptide probes to proteins containing suitable peptide substrates (seeref: Nat. Chem. Biol. 2007 November; 3(11):707-8. DOI:10.1038/nchembio.2007.31). The FLAG-tag (DYKDDDDK (SEQ ID NO: 2)),V5-tag (GKPIPNPLLGLDST (SEQ ID NO: 3)), GCN4-tag (EELLSKNYHLENEVARLKK(SEQ ID NO: 4)), HA-tag (YPYDVPDYA (SEQ ID NO: 5)), and Myc-tag(EQKLISEEDL (SEQ ID NO: 6)) are commonly employed as epitope tags forimmunoassays. The pi-clamp encodes a peptide sequence (FCPF (SEQ ID NO:622)) that can by labeled with a pentafluoro-aromatic substrates (ref:Nat. Chem. 2016 February; 8(2):120-8. doi: 10.1038/nchem.2413).

FIG. 26A shows precise installation of a His₆-tag and a FLAG-tag intogenomic DNA. A guide RNA targeting the HEK3 locus was designed with areverse transcription template that encodes either an 18-nt His-taginsertion or a 24-nt FLAG-tag insertion. Editing efficiency intransfected HEK cells was assessed using amplicon sequencing. Note thatthe full 24-nt sequence of the FLAG-tag is outside of the viewing frame(sequencing confirmed full and precise insertion). FIG. 26B shows aschematic outlining various applications involving protein/peptidetagging, including (a) rendering proteins soluble or insoluble, (b)changing or tracking the cellular localization of a protein, (c)extending the half-life of a protein, (d) facilitating proteinpurification, and (e) facilitating the detection of proteins.

FIG. 27 shows an overview of prime editing by installing a protectivemutation in PRNP that prevents or halts the progression of priondisease. The PEgRNA sequences correspond to SEQ ID NO: 4082 on the left(i.e., 5′ of the sgRNA scaffold) and SEQ ID NO 4083 on the right (i.e.,3′ of the sgRNA scaffold).

FIG. 28A is a schematic of PE-based insertion of sequences encoding RNAmotifs. FIG. 28B is a list (not exhaustive) of some example motifs thatcould potentially be inserted, and their functions.

FIG. 29A is a depiction of a prime editor. FIG. 29B shows possiblemodifications to genomic, plasmid, or viral DNA directed by a PE. FIG.29C shows an example scheme for insertion of a library of peptide loopsinto a specified protein (in this case GFP) via a library of PEgRNAs.FIG. 29D shows an example of possible programmable deletions of codonsor N-, or C-terminal truncations of a protein using different PEgRNAs.Deletions would be predicted to occur with minimal generation offrameshift mutations.

FIG. 30 shows a possible scheme for iterative insertion of codons in acontinual evolution system, such as PACE.

FIG. 31 is an illustration of an engineered gRNA showing the gRNA core,˜20nt spacer matching the sequence of the targeted gene, the reversetranscription template with immunogenic epitope nucleotide sequence andthe primer binding site matching the sequence of the targeted gene.

FIG. 32 is a schematic showing using prime editing as a means to insertknown immunogenicity epitopes into endogenous or foreign genomic DNA,resulting in modification of the corresponding proteins.

FIG. 33 is a schematic showing PEgRNA design for primer binding sequenceinsertions and primer binding insertion into genomic DNA using primeediting for determining off-target editing. In this embodiment, primeediting is conducted inside a living cell, a tissue, or an animal model.As a first step, an appropriate PEgRNA is designed. The top schematicshows an exemplary PEgRNA that may be used in this aspect. The spacer inthe PEgRNA (labeled “protospacer”) is complementary to one of thestrands of the genomic target. The PE:PEgRNA complex (i.e., the PEcomplex) installs a single stranded 3′ end flap at the nick site whichcontains the encoded primer binding sequence and the region of homology(coded by the homology arm of the PEgRNA) that is complementary to theregion just downstream of the cut site (in bold). Through flap invasionand DNA repair/replication processes, the synthesized strand becomesincorporated into the DNA, thereby installing the primer binding site.This process can occur at the desired genomic target, but also at othergenomic sites that might interact with the PEgRNA in an off-targetmanner (i.e., the PEgRNA guides the PE complex to other off-target sitesdue to the complementarity of the spacer region to other genomic sitesthat are not the intended genomic site). Thus, the primer bindingsequence may be installed not only at the desired genomic target, but atoff-target genomic sites elsewhere in the genome. In order to detect theinsertion of these primer binding sites at both the intended genomictarget sites and the off-target genomic sites, the genomic DNA (post-PE)can be isolated, fragmented, and ligated to adapter nucleotides(striped). Next, PCR may be carried out with PCR oligonucleotides thatanneal to the adapters and to the inserted primer binding sequence toamplify on-target and off-target genomic DNA regions into which theprimer binding site was inserted by PE. High throughput sequencing thenme be conducted to and sequence alignments to identify the insertionpoints of PE-inserted primer binding sequences at either the on-targetsite or at off-target sites.

FIG. 34 is a schematic showing the precise insertion of a gene with PE.

FIG. 35A is a schematic showing the natural insulin signaling pathway.FIG. 35B is a schematic showing FKBP12-tagged insulin receptoractivation controlled by FK1012.

FIG. 36 shows small-molecule monomers. References: bumped FK506 mimic(2)¹⁰⁷

FIGS. 37A-37B show small-molecule dimers. References: FK1012 4^(95,96);FK1012 5¹⁰⁸; FK1012 6¹⁰⁷; AP1903 7¹⁰⁷; cyclosporin A dimer 8⁹⁸;FK506-cyclosporin A dimer (FkCsA) 9¹⁰⁰.

FIGS. 38A-38F provide an overview of prime editing and feasibilitystudies in vitro and in yeast cells. FIG. 38A shows the 75,122 knownpathogenic human genetic variants in ClinVar (accessed July, 2019),classified by type. FIG. 38B shows that a prime editing complex consistsof a prime editor (PE) protein containing an RNA-guided DNA-nickingdomain, such as Cas9 nickase, fused to an engineered reversetranscriptase domain and complexed with a prime editing guide RNA(PEgRNA). The PE:PEgRNA complex binds the target DNA site and enables alarge variety of precise DNA edits at a wide range of DNA positionsbefore or after the target site's protospacer adjacent motif (PAM). FIG.38C shows that upon DNA target binding, the PE:PEgRNA complex nicks thePAM-containing DNA strand. The resulting free 3′ end hybridizes to theprimer-binding site of the PEgRNA. The reverse transcriptase domaincatalyzes primer extension using the RT template of the PEgRNA,resulting in a newly synthesized DNA strand containing the desired edit(the 3′ flap).Equilibration between the edited 3′ flap and the unedited5′ flap containing the original DNA, followed by cellular 5′ flapcleavage and ligation, and DNA repair or replication to resolve theheteroduplex DNA, results in stably edited DNA. FIG. 38D shows in vitro5′-extended PEgRNA primer extension assays with pre-nicked dsDNAsubstrates containing 5′-Cy5 labeled PAM strands, dCas9, and acommercial M-MLV RT variant (RT, Superscript III). dCas9 was complexedwith PEgRNAs containing RT template of varying lengths, then added toDNA substrates along with the indicated components. Reactions wereincubated at 37° C. for 1 hour, then analyzed by denaturing urea PAGEand visualized for Cy5 fluorescence. FIG. 38E shows primer extensionassays performed as in FIG. 38D using 3′-extended PEgRNAs pre-complexedwith dCas9 or Cas9 H840A nickase, and pre-nicked or non-nicked5′-Cy5-labeled dsDNA substrates. FIG. 38F shows yeast coloniestransformed with GFP-mCherry fusion reporter plasmids edited in vitrowith PEgRNAs, Cas9 nickase, and RT. Plasmids containing nonsense orframeshift mutations between GFP and mCherry were edited with5′-extended or 3′-extended PEgRNAs that restore mCherry translation viatransversion mutation, 1-bp insertion, or 1-bp deletion. GFP and mCherrydouble-positive cells reflect successful editing.

FIGS. 39A-39D show prime editing of genomic DNA in human cells by PE1and PE2. FIG. 39A shows PEgRNAs contain a spacer sequence, a sgRNAscaffold, and a 3′ extension containing a primer-binding site (bold,underlined italics) and a reverse transcription (RT) template (bolditalics), which contains the edited base(s) (bold). The primer-bindingsite hybridizes to the PAM-containing DNA strand immediately upstream ofthe site of nicking. The RT template is homologous to the DNA sequencedownstream of the nick, with the exception of the encoded edit. FIG. 39Bshows an installation of a T·A-to-A·T transversion edit at the HEK3 sitein HEK293T cells using Cas9 H840A nickase fused to wild-type M-MLVreverse transcriptase (PE1) and PEgRNAs of varying primer-binding sitelengths. FIG. 39C shows the use of an engineered pentamutant M-MLVreverse transcriptase (D200N, L603W, T306K, W313F, T330P) in PE2substantially improves prime editing transversion efficiencies at fivegenomic sites in HEK293T cells, and small insertion and small deletionedits at HEK3. FIG. 39D is a comparison of PE2 editing efficiencies withvarying RT template lengths at five genomic sites in HEK293T cells.Values and error bars reflect the mean and s.d. of three independentbiological replicates.

FIGS. 40A-40C show PE3 and PE3b systems nick the non-edited strand toincrease prime editing efficiency. FIG. 40A is an overview of the primeediting by PE3. After initial synthesis of the edited strand, DNA repairwill remove either the newly synthesized strand containing the edit (3′flap excision) or the original genomic DNA strand (5′ flap excision). 5′flap excision leaves behind a DNA heteroduplex containing one editedstrand and one non-edited strand. Mismatch repair machinery or DNAreplication could resolve the heteroduplex to give either edited ornon-edited products. Nicking the non-edited strand favors repair of thatstrand, resulting in preferential generation of stable duplex DNAcontaining the desired edit. FIG. 40B shows the effect of complementarystrand nicking on PE3-mediated prime editing efficiency and indelformation. “None” refers to PE2 controls, which do not nick thecomplementary strand. FIG. 40C is a comparison of editing efficiencieswith PE2 (no complementary strand nick), PE3 (general complementarystrand nick), and PE3b (edit-specific complementary strand nick). Allediting yields reflect the percentage of total sequencing reads thatcontain the intended edit and do not contain indels among all treatedcells, with no sorting. Values and error bars reflect the mean and s.d.of three independent biological replicates.

FIGS. 41A-41K show targeted insertions, deletions, and all 12 types ofpoint mutations with PE3 at seven endogenous human genomic loci inHEK293T cells. FIG. 41A is a graph showing all 12 types ofsingle-nucleotide transition and transversion edits from position +1 to+8 (counting the location of the PEgRNA-induced nick as between position+1 and −1) of the HEK3 site using a 10-nt RT template. FIG. 41B is agraph showing long-range PE3 transversion edits at the HEK3 site using a34-nt RT template. FIGS. 41C-41H are graphs showing all 12 types oftransition and transversion edits at various positions in the primeediting window for (FIG. 41C) RNF2, (FIG. 41D) FANCF, (FIG. 41E) EMX1,(FIG. 41F) RUNX1, (FIG. 41G) VEGFA, and (FIG. 41H) DNMT1. FIG. 41I is agraph showing targeted 1- and 3-bp insertions, and 1- and 3-bp deletionswith PE3 at seven endogenous genomic loci. FIG. 41J is a graph showingthe targeted precise deletions of 5 to 80 bp at the HEK3 target site.FIG. 41K is a graph showing a combination edits of insertions anddeletions, insertions and point mutations, deletions and pointmutations, and double point mutations at three endogenous genomic loci.All editing yields reflect the percentage of total sequencing reads thatcontain the intended edit and do not contain indels among all treatedcells, with no sorting. Values and error bars reflect the mean and s.d.of three independent biological replicates.

FIGS. 42A-42H show the comparison of prime editing and base editing, andoff-target editing by Cas9 and PE3 at known Cas9 off-target sites. FIG.42A shows total C·G-to-T·A editing efficiency at the same targetnucleotides for PE2, PE3, BE2max, and BE4max at endogenous HEK3, FANCF,and EMX1 sites in HEK293T cells. FIG. 42B shows indel frequency fromtreatments in FIG. 42A. FIG. 42C shows the editing efficiency of preciseC·G-to-T·A edits (without bystander edits or indels) for PE2, PE3,BE2max, and BE4max at HEK3, FANCF, and EMX1. For EMX1, precise PEcombination edits of all possible combinations of C·G-to-T·A conversionat the three targeted nucleotides are also shown.

FIG. 42D shows the total A·T-to-G·C editing efficiency for PE2, PE3,ABEdmax, and ABEmax at HEK3 and FANCF. FIG. 42E shows the preciseA·T-to-G·C editing efficiency without bystander edits or indels for atHEK3 and FANCF. FIG. 42F shows indel frequency from treatments in FIG.42D. FIG. 42G shows the average triplicate editing efficiencies(percentage sequencing reads with indels) in HEK293T cells for Cas9nuclease at four on-target and 16 known off-target sites. The 16off-target sites examined were the top four previously reportedoff-target sites^(118,159) for each of the four on-target sites. Foreach on-target site, Cas9 was paired with a sgRNA or with each of fourPEgRNAs that recognize the same protospacer. FIG. 42H shows the averagetriplicate on-target and off-target editing efficiencies and indelefficiencies (below in parentheses) in HEK293T cells for PE2 or PE3paired with each PEgRNA in (FIG. 42G). On-target editing yields reflectthe percentage of total sequencing reads that contain the intended editand do not contain indels among all treated cells, with no sorting.Off-target editing yields reflect off-target locus modificationconsistent with prime editing. Values and error bars reflect the meanand s.d. of three independent biological replicates.

FIGS. 43A-43I show prime editing in various human cell lines and primarymouse cortical neurons, installation and correction of pathogenictransversion, insertion, or deletion mutations, and comparison of primeediting and HDR. FIG. 43A is a graph showing the installation (viaT·A-to-A·T transversion) and correction (via A·T-to-T·A transversion) ofthe pathogenic E6V mutation in HBB in HEK293T cells. Correction eitherto wild-type HBB, or to HBB containing a silent mutation that disruptsthe PEgRNA PAM, is shown. FIG. 43B is a graph showing the installation(via 4-bp insertion) and correction (via 4-bp deletion) of thepathogenic HEXA 1278+TATC allele in HEK293T cells. Correction either towild-type HEXA, or to HEXA containing a silent mutation that disruptsthe PEgRNA PAM, is shown.

FIG. 43C is a graph showing the installation of the protective G127Vvariant in PRNP in HEK293T cells via G·C-to-T·A transversion. FIG. 43Dis a graph showing prime editing in other human cell lines includingK562 (leukemic bone marrow cells), U2OS (osteosarcoma cells), and HeLa(cervical cancer cells). FIG. 43E is a graph showing the installation ofa G·C-to-T·A transversion mutation in DNMT1 of mouse primary corticalneurons using a dual split-intein PE3 lentivirus system, in which theN-terminal half is Cas9 (1-573) fused to N-intein and through a P2Aself-cleaving peptide to GFP-KASH, and the C-terminal half is theC-intein fused to the remainder of PE2. PE2 halves are expressed from ahuman synapsin promoter that is highly specific for mature neurons.Sorted values reflect editing or indels from GFP-positive nuclei, whileunsorted values are from all nuclei. FIG. 43F is a comparison of PE3 andCas9-mediated HDR editing efficiencies at endogenous genomic loci inHEK293T cells. FIG. 43G is a comparison of PE3 and Cas9-mediated HDRediting efficiencies at endogenous genomic loci in K562, U2OS, and HeLacells. FIG. 43H is a comparison of PE3 and Cas9-mediated HDR indelbyproduct generation in HEK293T, K562, U2OS, and HeLa cells. FIG. 43Ishows targeted insertion of a His6 tag (18 bp), FLAG epitope tag (24bp), or extended LoxP site (44 bp) in HEK293T cells by PE3. All editingyields reflect the percentage of total sequencing reads that contain theintended edit and do not contain indels among all treated cells. Valuesand error bars reflect the mean and s.d. of three independent biologicalreplicates.

FIGS. 44A-44G show in vitro prime editing validation studies withfluorescently labeled DNA substrates. FIG. 4A shows electrophoreticmobility shift assays with dCas9, 5′-extended PEgRNAs and 5′-Cy5-labeledDNA substrates. PEgRNAs 1 through 5 contain a 15-nt linker sequence(linker A for PEgRNA 1, linker B for PEgRNAs 2 through 5) between thespacer and the PBS, a 5-nt PBS sequence, and RT templates of 7 nt(PEgRNAs 1 and 2), 8 nt (PEgRNA 3), 15 nt (PEgRNA 4), and 22 nt (PEgRNA5). PEgRNAs are those used in FIGS. 44E and 44F; full sequences arelisted in Tables 2A-2C. FIG. 44B shows in vitro nicking assays of Cas9H840A using 5′-extended and 3′-extended PEgRNAs. FIG. 44C showsCas9-mediated indel formation in HEK293T cells at HEK3 using 5′-extendedand 3′-extended PEgRNAs. FIG. 44D shows an overview of prime editing invitro biochemical assays. 5′-Cy5-labeled pre-nicked and non-nicked dsDNAsubstrates were tested. sgRNAs, 5′-extended PEgRNAs, or 3′-extendedPEgRNAs were pre-complexed with dCas9 or Cas9 H840A nickase, thencombined with dsDNA substrate, M-MLV RT, and dNTPs. Reactions wereallowed to proceed at 37° C. for 1 hour prior to separation bydenaturing urea PAGE and visualization by Cy5 fluorescence. FIG. 44Eshows primer extension reactions using 5′-extended PEgRNAs, pre-nickedDNA substrates, and dCas9 lead to significant conversion to RT products.FIG. 44F shows primer extension reactions using 5′-extended PEgRNAs asin FIG. 44B, with non-nicked DNA substrate and Cas9 H840A nickase.Product yields are greatly reduced by comparison to pre-nickedsubstrate. FIG. 44G shows an in vitro primer extension reaction using a3′-PEgRNA generates a single apparent product by denaturing urea PAGE.The RT product band was excised, eluted from the gel, then subjected tohomopolymer tailing with terminal transferase (TdT) using either dGTP ordATP. Tailed products were extended by poly-T or poly-C primers, and theresulting DNA was sequenced. Sanger traces indicate that threenucleotides derived from the gRNA scaffold were reverse transcribed(added as the final 3′ nucleotides to the DNA product). Note that inmammalian cell prime editing experiments, PEgRNA scaffold insertion ismuch rarer than in vitro (FIGS. 56A-56D), potentially due to theinability of the tethered reverse transcriptase to access the Cas9-boundguide RNA scaffold, and/or cellular excision of mismatched 3′ ends of 3′flaps containing PEgRNA scaffold sequences.

FIGS. 45A-45G show cellular repair in yeast of 3′ DNA flaps from invitro prime editing reactions. FIG. 45A shows that dual fluorescentprotein reporter plasmids contain GFP and mCherry open reading framesseparated by a target site encoding an in-frame stop codon, a +1frameshift, or a −1 frameshift. Prime editing reactions were carried outin vitro with Cas9 H840A nickase, PEgRNA, dNTPs, and M-MLV reversetranscriptase, and then transformed into yeast. Colonies that containunedited plasmids produce GFP but not mCherry. Yeast colonies containingedited plasmids produce both GFP and mCherry as a fusion protein. FIG.45B shows an overlay of GFP and mCherry fluorescence for yeast coloniestransformed with reporter plasmids containing a stop codon between GFPand mCherry (unedited negative control, top), or containing no stopcodon or frameshift between GFP and mCherry (pre-edited positivecontrol, bottom). FIGS. 45C-45F show a visualization of mCherry and GFPfluorescence from yeast colonies transformed with in vitro prime editingreaction products. FIG. 45C shows a stop codon correction via T·A-to-A·Ttransversion using a 3′-extended PEgRNA, or a 5′-extended PEgRNA, asshown in FIG. 45D. FIG. 45E shows a +1 frameshift correction via a 1-bpdeletion using a 3′-extended PEgRNA. FIG. 45F shows a −1 frameshiftcorrection via a 1-bp insertion using a 3′-extended PEgRNA. FIG. 45Gshows Sanger DNA sequencing traces from plasmids isolated from GFP-onlycolonies in FIG. 45B and GFP and mCherry double-positive colonies inFIG. 45C.

FIGS. 46A-46F show correct editing versus indel generation with PE1.FIG. 46A shows T·A-to-A·T transversion editing efficiency and indelgeneration by PE1 at the +1 position of HEK3 using PEgRNAs containing10-nt RT templates and a PBS sequences ranging from 8-17 nt. FIG. 46Bshows G·C-to-T·A transversion editing efficiency and indel generation byPE1 at the +5 position of EMX1 using PEgRNAs containing 13-nt RTtemplates and a PBS sequences ranging from 9-17 nt. FIG. 46C showsG·C-to-T·A transversion editing efficiency and indel generation by PE1at the +5 position of FANCF using PEgRNAs containing 17-nt RT templatesand a PBS sequences ranging from 8-17 nt. FIG. 46D shows C·G-to-A·Ttransversion editing efficiency and indel generation by PE1 at the +1position of RNF2 using PEgRNAs containing 11-nt RT templates and a PBSsequences ranging from 9-17 nt. FIG. 46E shows G·C-to-T·A transversionediting efficiency and indel generation by PE1 at the +2 position ofHEK4 using PEgRNAs containing 13-nt RT templates and a PBS sequencesranging from 7-15 nt. FIG. 46F shows PE1-mediated +1 T deletion, +1 Ainsertion, and +1 CTT insertion at the HEK3 site using a 13-nt PBS and10-nt RT template. Sequences of PEgRNAs are those used in FIG. 39C (seeTables 3A-3R). Values and error bars reflect the mean and s.d. of threeindependent biological replicates.

FIGS. 47A-47S show the evaluation of M-MLV RT variants for primeediting. FIG. 47A shows the abbreviations for prime editor variants usedin this figure. FIG. 47B shows targeted insertion and deletion editswith PE1 at the HEK3 locus. FIGS. 47C-47H show a comparison of 18 primeeditor constructs containing M-MLV RT variants for their ability toinstall a +2 G·C-to-C·G transversion edit at HEK3 as shown in FIG. 47C,a 24-bp FLAG insertion at HEK3 as shown in FIG. 47D, a +1 C·G-to-A·Ttransversion edit at RNF2 as shown in FIG. 47E, a +1 G·C-to-C·Gtransversion edit at EMX1 as shown in FIG. 47F, a +2 T·A-to-A·Ttransversion edit at HBB as shown in FIG. 47G, and a +1 G·C-to-C·Gtransversion edit at FANCF as shown in FIG. 47H. FIGS. 47I-47N show acomparison of four prime editor constructs containing M-MLV variants fortheir ability to install the edits shown in FIGS. 47C-47H in a secondround of independent experiments. FIGS. 47O-47S show PE2 editingefficiency at five genomic loci with varying PBS lengths. FIG. 47O showsa +1 T·A-to-A·T variation at HEK3. FIG. 47P shows a +5 G·C-to-T·Avariation at EMX1. FIG. 47Q shows a +5 G·C-to-T·A variation at FANCF.FIG. 47R shows a +1 C·G-to-A·T variation at RNF2. FIG. 47S shows a +2G·C-to-T·A variation at HEK4. Values and error bars reflect the mean ands.d. of three independent biological replicates.

FIGS. 48A-48C show design features of PEgRNA PBS and RT templatesequences. FIG. 48A shows PE2-mediated +5 G·C-to-T·A transversionediting efficiency (solid line) at VEGFA in HEK293T cells as a functionof RT template length. Indels (dotted line) are plotted for comparison.The sequence below the graph shows the last nucleotide templated forsynthesis by the PEgRNA. G nucleotides (templated by a C in the PEgRNA)are highlighted; RT templates that end in C should be avoided duringPEgRNA design to maximize prime editing efficiencies. FIG. 48B shows +5G·C-to-T·A transversion editing and indels for DNMT1 as in FIG. 48A.FIG. 48C shows +5 G·C-to-T·A transversion editing and indels for RUNX1as in FIG. 48A. Values and error bars reflect the mean and s.d. of threeindependent biological replicates.

FIGS. 49A-49B show the effects of PE2, PE2 R110S K103L, Cas9 H840Anickase, and dCas9 on cell viability. HEK293T cells were transfectedwith plasmids encoding PE2, PE2 R110S K103L, Cas9 H840A nickase, ordCas9, together with a HEK3-targeting PEgRNA plasmid. Cell viability wasmeasured every 24 hours post-transfection for 3 days using theCellTiter-Glo 2.0 assay (Promega). FIG. 49A shows viability, as measuredby luminescence, at 1, 2, or 3 days post-transfection. Values and errorbars reflect the mean and s.e.m. of three independent biologicalreplicates each performed in technical triplicate. FIG. 49B showspercent editing and indels for PE2, PE2 R110S K103L, Cas9 H840A nickase,or dCas9, together with a HEK3-targeting PEgRNA plasmid that encodes a+5 G to A edit. Editing efficiencies were measured on day 3post-transfection from cells treated alongside of those used forassaying viability in FIG. 49A. Values and error bars reflect the meanand s.d. of three independent biological replicates.

FIGS. 50A-50B show PE3-mediated HBB E6V correction and HEXA 1278+TATCcorrection by various PEgRNAs. FIG. 50A shows a screen of 14 PEgRNAs forcorrection of the HBB E6V allele in HEK293T cells with PE3. All PEgRNAsevaluated convert the HBB E6V allele back to wild-type HBB without theintroduction of any silent PAM mutation. FIG. 50B shows a screen of 41PEgRNAs for correction of the HEXA 1278+TATC allele in HEK293T cellswith PE3 or PE3b. Those PEgRNAs labeled HEXAs correct the pathogenicallele by a shifted 4-bp deletion that disrupts the PAM and leaves asilent mutation. Those PEgRNAs labeled HEXA correct the pathogenicallele back to wild-type. Entries ending in “b” use an edit-specificnicking sgRNA in combination with the PEgRNA (the PE3b system). Valuesand error bars reflect the mean and s.d. of three independent biologicalreplicates.

FIGS. 51A-51G show a PE3 activity in human cell lines and a comparisonof PE3 and Cas9-initiated HDR. Efficiency of generating the correct edit(without indels) and indel frequency for PE3 and Cas9-initiated HDR inHEK293T cells as shown in FIG. 51A, K562 cells as shown in FIG. 51B,U2OS cells as shown in FIG. 51C, and HeLa cells as shown in FIG. 51D.Each bracketed editing comparison installs identical edits with PE3 andCas9-initiated HDR. Non-targeting controls are PE3 and a PEgRNA thattargets a non-target locus. FIG. 51E shows control experiments withnon-targeting PEgRNA+PE3, and with dCas9+sgRNA, compared with wild-typeCas9 HDR experiments confirming that ssDNA donor HDR template, a commoncontaminant that artificially elevates apparent HDR efficiencies, doesnot contribute to the HDR measurements in FIGS. 51A-51D. FIGS. 51F-51Gshow example HEK3 site allele tables from genomic DNA samples isolatedfrom K562 cells after editing with PE3 or with Cas9-initiated HDR.Alleles were sequenced on an Illumina MiSeq and analyzed withCRISPResso2¹⁷⁸. The reference HEK3 sequence from this region is at thetop. Allele tables are shown for a non-targeting PEgRNA negativecontrol, a +1 CTT insertion at HEK3 using PE3, and a +1 CTT insertion atHEK3 using Cas9-initiated HDR. Allele frequencies and correspondingIllumina sequencing read counts are shown for each allele. All allelesobserved with frequency ≥0.20% are shown. Values and error bars reflectthe mean and s.d. of three independent biological replicates.

FIGS. 52A-52D show distribution by length of pathogenic insertions,duplications, deletions, and indels in the ClinVar database. The ClinVarvariant summary was downloaded from NCBI Jul. 15, 2019. The lengths ofreported insertions, deletions, and duplications were calculated usingreference and alternate alleles, variant start and stop positions, orappropriate identifying information in the variant name. Variants thatdid not report any of the above information were excluded from theanalysis. The lengths of reported indels (single variants that includeboth insertions and deletions relative to the reference genome) werecalculated by determining the number of mismatches or gaps in the bestpairwise alignment between the reference and alternate alleles.

FIGS. 53A-53E show FACS gating examples for GFP-positive cell sorting.Below are examples of original batch analysis files outlining thesorting strategy used for generating HEXA 1278+TATC and HBB E6V HEK293Tcell lines. The image data was generated on a Sony LE-MA900 cytometerusing Cell Sorter Software v. 3.0.5. Graphic 1 shows gating plots forcells that do not express GFP. Graphic 2 shows an example sort ofP2A-GFP-expressing cells used for isolating the HBB E6V HEK293T celllines. HEK293T cells were initially gated on population usingFSC-A/BSC-A (Gate A), then sorted for singlets using FSC-A/FSC-H (GateB). Live cells were sorted for by gating DAPI-negative cells (Gate C).Cells with GFP fluorescence levels that were above those of thenegative-control cells were sorted for using EGFP as the fluorochrome(Gate D). FIG. 53A shows HEK293T cells (GFP-negative). FIG. 53B shows arepresentative plot of FACS gating for cells expressing PE2-P2A-GFP.FIG. 53C shows the genotypes for HEXA 1278+TATC homozygote HEK293Tcells. FIGS. 53D-53E show allele tables for HBB E6V homozygote HEK293Tcell lines.

FIG. 54 is a schematic which summarizes the PEgRNA cloning procedure.

FIGS. 55A-55G are schematics of PEgRNA designs. FIG. 55A shows a simplediagram of PEgRNA with domains labeled (left) and bound to nCas9 at agenomic site (right). FIG. 55B shows various types of modifications toPEgRNA which are anticipated to increase activity. FIG. 55C showsmodifications to PEgRNA to increase transcription of longer RNAs viapromoter choice and 5′, 3′ processing and termination. FIG. 55D showsthe lengthening of the P1 system, which is an example of a scaffoldmodification. FIG. 55E shows that the incorporation of syntheticmodifications within the template region, or elsewhere within thePEgRNA, could increase activity. FIG. 55F shows that a designedincorporation of minimal secondary structure within the template couldprevent formation of longer, more inhibitory, secondary structure. FIG.55G shows a split PEgRNA with a second template sequence anchored by anRNA element at the 3′ end of the PEgRNA (left). Incorporation ofelements at the 5′ or 3′ ends of the PEgRNA could enhance RT binding.

FIGS. 56A-56D show the incorporation of PEgRNA scaffold sequence intotarget loci. HTS data were analyzed for PEgRNA scaffold sequenceinsertion as described in FIGS. 60A-60B. FIG. 56A shows an analysis forthe EMX1 locus. Shown is the % of total sequencing reads containing oneor more PEgRNA scaffold sequence nucleotides within an insertionadjacent to the RT template (left); the percentage of total sequencingreads containing a PEgRNA scaffold sequence insertion of the specifiedlength (middle); and the cumulative total percentage of PEgRNA insertionup to and including the length specified on the X axis. FIG. 56B showsthe same as FIG. 56A, but for FANCF. FIG. 56C shows the same as in FIG.56A but for HEK3. FIG. 56D shows the same as FIG. 56A but for RNF2.Values and error bars reflect the mean and s.d. of three independentbiological replicates.

FIGS. 57A-57I show the effects of PE2, PE2-dRT, and Cas9 H840A nickaseon transcriptome-wide RNA abundance. Analysis of cellular RNA, depletedfor ribosomal RNA, isolated from HEK293T cells expressing PE2, PE2-dRT,or Cas9 H840A nickase and a PRNP-targeting or HEXA-targeting PEgRNA.RNAs corresponding to 14,410 genes and 14,368 genes were detected inPRNP and HEXA samples, respectively. FIGS. 57A-57F show Volcano plotdisplaying the −log 10 FDR-adjusted p-value vs. log 2-fold change intranscript abundance for Aeach RNA, comparing (FIG. 57A) PE2 vs. PE2-dRTwith PRNP-targeting PEgRNA, (FIG. 57B) PE2 vs. Cas9 H840A withPRNP-targeting PEgRNA, (FIG. 57C) PE2-dRT vs. Cas9 H840A withPRNP-targeting PEgRNA, (FIG. 57D) PE2 vs. PE2-dRT with HEXA-targetingPEgRNA, (FIG. 57E) PE2 vs. Cas9 H840A with HEXA-targeting PEgRNA, (FIG.57F) PE2-dRT vs. Cas9 H840A with HEXA-targeting PEgRNA. Gray dotsindicate genes that show ≥2-fold change in relative abundance that arestatistically significant (FDR-adjusted p<0.05). FIGS. 57G-57I are Venndiagrams of upregulated and downregulated transcripts (≥2-fold change)comparing PRNP and HEXA samples for (FIG. 57G) PE2 vs PE2-dRT, (FIG.57H) PE2 vs. Cas9 H840A, and (FIG. 57I) PE2-dRT vs. Cas9 H840A.

FIGS. 58A-58B show representative FACS gating for neuronal nucleisorting. Nuclei were sequentially gated on the basis of DyeCycle Rubysignal, FSC/SSC ratio, SSC-Width/SSC-height ratio, and GFP/DyeCycleratio.

FIGS. 59A-59G show the protocol for cloning 3′-extended PEgRNAs intomammalian U6 expression vectors by Golden Gate assembly. FIG. 59A showsthe cloning overview. FIG. 59B shows ‘Step 1: DigestpU6-PEgRNA-GG-Vector plasmid (component 1)’. FIG. 59C shows ‘Steps 2 and3: Order and anneal oligonucleotide parts (components 2, 3, and 4)’.FIG. 59D shows ‘Step 2.b.ii.: sgRNA scaffold phosphorylation(unnecessary if oligonucleotides were purchased phosphorylated)’. FIG.59E shows ‘Step 4: PEgRNA assembly’. FIG. 59F shows ‘Steps 5 and 6:Transformation of assembled plasmids’. FIG. 59G shows a diagramsummarizing the PEgRNA cloning protocol.

FIGS. 60A-60B show the Python script for quantifying PEgRNA scaffoldintegration. A custom python script was generated to characterize andquantify PEgRNA insertions at target genomic loci. The scriptiteratively matches text strings of increasing length taken from areference sequence (guide RNA scaffold sequence) to the sequencing readswithin fastq files, and counts the number of sequencing reads that matchthe search query. Each successive text string corresponds to anadditional nucleotide of the guide RNA scaffold sequence. Exact lengthintegrations and cumulative integrations up to a specified length werecalculated in this manner. At the start of the reference sequence, 5 to6 bases of the 3′ end of the new DNA strand synthesized by the reversetranscriptase are included to ensure alignment and accurate counting ofshort slices of the sgRNA.

FIG. 61 is a graph showing the percent of total sequencing reads withthe specified edit for SaCas9(N580A)-MMLV RT HEK3+6 C>A. The values forthe correct edits as well as indels are shown.

FIGS. 62A-62B show the importance of the protospacer for efficientinstallation of a desired edit at a precise location with prime editing.FIG. 62A is a graph showing the percent of total sequencing reads withtarget T·A base pairs converted to A·T for various HEK3 loci. FIG. 62Bis a sequence analysis showing the same.

FIG. 63 is a graph showing SpCas9 PAM variants in PAM editing (N=3). Thepercent of total sequencing reads with the targeted PAM edit is shownfor SpCas9(H840A)-VRQR-MMLV RT, where NGA >NTA, and forSpCas9(H840A)-VRER-MMLV RT, where NGCG >NTCG. The PEgRNA primer bindingsite (PBS) length, RT template (RT) length, and PE system used arelisted.

FIGS. 64A-64F depict a schematic showing the introduction of varioussite-specific recombinase (SSR) targets into the genome using PE. FIG.64A provides a general schematic of the insertion of a recombinasetarget sequence by a prime editor. FIG. 64B shows how a single SSRtarget inserted by PE can be used as a site for genomic integration of aDNA donor template. FIG. 64C shows how a tandem insertion of SSR targetsites can be used to delete a portion of the genome. FIG. 64D shows howa tandem insertion of SSR target sites can be used to invert a portionof the genome. FIG. 64E shows how the insertion of two SSR target sitesat two distal chromosomal regions can result in chromosomaltranslocation. FIG. 64F shows how the insertion of two different SSRtarget sites in the genome can be used to exchange a cassette from a DNAdonor template. See Example 17 for further details.

FIG. 65 shows in 1) the PE-mediated synthesis of a SSR target site in ahuman cell genome and 2) the use of that SSR target site to integrate aDNA donor template comprising a GFP expression marker. Once successfullyintegrated, the GFP causes the cell to fluoresce. See Example 17 forfurther details.

FIG. 66 depicts one embodiment of a prime editor being provided as twoPE half proteins which regenerate as whole prime editor through theself-splicing action of the split-intein halves located at the end orbeginning of each of the prime editor half proteins.

FIGS. 67A-67B depict the mechanism of intein removal from a polypeptidesequence and the reformation of a peptide bond between the N-terminaland the C-terminal extein sequences. FIG. 67A depicts the generalmechanism of two half proteins each containing half of an inteinsequence, which when in contact within a cell result in afully-functional intein which then undergoes self-spicing and excision.The process of excision results in the formation of a peptide bondbetween the N-terminal protein half (or the “N extein”) and theC-terminal protein half (or the “C extein”) to form a whole, singlepolypeptide comprising the N extein and the C extein portions. Invarious embodiments, the N extein may correspond to the N-terminal halfof a split prime editor fusion protein and the C extein may correspondto the C-terminal half of a split prime editor. FIG. 67B shows achemical mechanism of intein excision and the reformation of a peptidebond that joins the N extein half (the bolded half) and the C exteinhalf (the thin-lined half). Excision of the split inteins (i.e., the Nintein and the C intein in the split intein configuration) may also bereferred to as “trans splicing” as it involves the splicing action oftwo separate components provided in trans.

FIG. 68A demonstrates that delivery of both split intein halves of SpPE(SEQ ID NO: 762) at the linker maintains activity at three test lociwhen co-transfected into HEK293T cells.

FIG. 68B demonstrates that delivery of both split intein halves of SaPE2(e.g., SEQ ID NO: 443 and SEQ ID NO: 450) recapitulate activity of fulllength SaPE2 (SEQ ID NO: 134) when co-transfected into HEK293T cells.Residues indicated in quotes are the sequence of amino acids 741-743 inSaCas9 (first residues of the C-terminal extein) which are important forthe intein trans splicing reaction. ‘SMP’ are the native residues, whichwe also mutated to the ‘CFN’ consensus splicing sequence. The consensussequence is shown to yield the highest reconstitution as measured byprime editing percentage.

FIG. 68C provides data showing that various disclosed PEribonucleoprotein complexes (PE2 at high concentration, PE3 at highconcentration and PE3 at low concentration) can be delivered in thismanner.

FIG. 69 shows a bacteriophage plaque assay to determine PE effectivenessin PANCE. Plaques (dark circles) indicate phage able to successfullyinfect E. coli. Increasing concentration of L-rhamnose results inincreased expression of PE and an increase in plaque formation.Sequencing of plaques revealed the presence of the PE-installed genomicedit.

FIGS. 70A-70I provide an example of an edited target sequence as anillustration of a step-by-step instruction for designing PEgRNAs andnicking-sgRNAs for prime editing. FIG. 70A: Step 1. Define the targetsequence and the edit. Retrieve the sequence of the target DNA region(˜200 bp) centered around the location of the desired edit (pointmutation, insertion, deletion, or combination thereof). FIG. 70B: Step2. Locate target PAMs. Identify PAMs in proximity to the edit location.Be sure to look for PAMs on both strands. While PAMs close to the editposition are preferred, it is possible to install edits usingprotospacers and PAMs that place the nick ≥30 nt from the edit position.FIG. 70C: Step 3. Locate the nick sites. For each PAM being considered,identify the corresponding nick site. For Sp Cas9 H840A nickase,cleavage occurs in the PAM-containing strand between the 3^(rd) and4^(th) bases 5′ to the NGG PAM. All edited nucleotides must exist 3′ ofthe nick site, so appropriate PAMs must place the nick 5′ to the targetedit on the PAM-containing strand. In the example shown below, there aretwo possible PAMs. For simplicity, the remaining steps will demonstratethe design of a PEgRNA using PAM 1 only. FIG. 70D: Step 4. Design thespacer sequence. The protospacer of Sp Cas9 corresponds to the 20nucleotides 5′ to the NGG PAM on the PAM-containing strand. EfficientPol III transcription initiation requires a G to be the firsttranscribed nucleotide. If the first nucleotide of the protospacer is aG, the spacer sequence for the PEgRNA is simply the protospacersequence. If the first nucleotide of the protospacer is not a G, thespacer sequence of the PEgRNA is G followed by the protospacer sequence.FIG. 70E: Step 5. Design a primer binding site (PBS). Using the startingallele sequence, identify the DNA primer on the PAM-containing strand.The 3′ end of the DNA primer is the nucleotide just upstream of the nicksite (i.e. the 4^(th) base 5′ to the NGG PAM for Sp Cas9). As a generaldesign principle for use with PE2 and PE3, a PEgRNA primer binding site(PBS) containing 12 to 13 nucleotides of complementarity to the DNAprimer can be used for sequences that contain ˜40-60% GC content. Forsequences with low GC content, longer (14- to 15-nt) PBSs should betested. For sequences with higher GC content, shorter (8- to 11-nt) PBSsshould be tested. Optimal PBS sequences should be determinedempirically, regardless of GC content. To design a length-p PBSsequence, take the reverse complement of the first p nucleotides 5′ ofthe nick site in the PAM-containing strand using the starting allelesequence. FIG. 70F: Step 6. Design an RT template. The RT templateencodes the designed edit and homology to the sequence adjacent to theedit. Optimal RT template lengths vary based on the target site. Forshort-range edits (positions +1 to +6), it is recommended to test ashort (9 to 12 nt), a medium (13 to 16 nt), and a long (17 to 20 nt) RTtemplate. For long-range edits (positions +7 and beyond), it isrecommended to use RT templates that extend at least 5 nt (preferably 10or more nt) past the position of the edit to allow for sufficient 3′ DNAflap homology. For long-range edits, several RT templates should bescreened to identify functional designs. For larger insertions anddeletions (>5 nt), incorporation of greater 3′ homology (˜20 nt or more)into the RT template is recommended. Editing efficiency is typicallyimpaired when the RT template encodes the synthesis of a G as the lastnucleotide in the reverse transcribed DNA product (corresponding to a Cin the RT template of the PEgRNA). As many RT templates supportefficient prime editing, avoidance of G as the final synthesizednucleotide is recommended when designing RT templates. To design alength-r RT template sequence, use the desired allele sequence and takethe reverse complement of the first r nucleotides 3′ of the nick site inthe strand that originally contained the PAM. Note that compared to SNPedits, insertion or deletion edits using RT templates of the same lengthwill not contain identical homology. FIG. 70G: Step 7. Assemble the fullPEgRNA sequence. Concatenate the PEgRNA components in the followingorder (5′ to 3′): spacer, scaffold, RT template and PBS. FIG. 70H: Step8. Designing nicking-sgRNAs for PE3. Identify PAMs on the non-editedstrand upstream and downstream of the edit. Optimal nicking positionsare highly locus-dependent and should be determined empirically. Ingeneral, nicks placed 40 to 90 nucleotides 5′ to the position acrossfrom the PEgRNA-induced nick lead to higher editing yields and fewerindels. A nicking sgRNA has a spacer sequence that matches the 20-ntprotospacer in the starting allele, with the addition of a 5′-G if theprotospacer does not begin with a G. FIG. 70I: Step 9. Designing PE3bnicking-sgRNAs. If a PAM exists in the complementary strand and itscorresponding protospacer overlaps with the sequence targeted forediting, this edit could be a candidate for the PE3b system. In the PE3bsystem, the spacer sequence of the nicking-sgRNA matches the sequence ofthe desired edited allele, but not the starting allele. The PE3b systemoperates efficiently when the edited nucleotide(s) falls within the seedregion (˜10 nt adjacent to the PAM) of the nicking-sgRNA protospacer.This prevents nicking of the complementary strand until afterinstallation of the edited strand, preventing competition between thePEgRNA and the sgRNA for binding the target DNA. PE3b also avoids thegeneration of simultaneous nicks on both strands, thus reducing indelformation significantly while maintaining high editing efficiency. PE3bsgRNAs should have a spacer sequence that matches the 20-nt protospacerin the desired allele, with the addition of a 5′ G if needed.

FIG. 71A shows the nucleotide sequence of a SpCas9 PEgRNA molecule (top)which terminates at the 3′ end in a “UUU” and does not contain a toeloopelement. The lower portion of the figure depicts the same SpCas9 PEgRNAmolecule but is further modified to contain a toeloop element having thesequence 5′-“GAAANNNNN”-3′ inserted immediately before the “UUU” 3′ end.The “N” can be any nucleobase.

FIG. 71B shows the results of Example 18, which demonstrates that theefficiency of prime editing in HEK cells or EMX cells is increased usingPEgRNA containing toeloop elements, whereas the percent of indelformation is largely unchanged.

FIGS. 72A-72C depict alternative PEgRNA configurations that can be usedin prime editing. FIG. 72A depicts the PE2:PEgRNA embodiment of primeediting. This embodiment involves a PE2 (a fusion protein comprising aCas9 and a reverse transcriptase) complexed with a PEgRNA (as alsodescribed in FIGS. 1A-II and/or FIGS. 3A-3E). In this embodiment, thetemplate for reverse transcription is incorporated into a 3′ extensionarm on the sgRNA to make the PEgRNA, and the DNA polymerase enzyme is areverse transcriptase (RT) fused directly to Cas9. FIG. 72B depicts theMS2cp-PE2:sgRNA+tPERT embodiment. This embodiment comprises a PE2 fusion(Cas9+a reverse transcriptase) that is further fused to the MS2bacteriophage coat protein (MS2cp) to form the MS2cp-PE2 fusion protein.To achieve prime editing, the MS2cp-PE2 fusion protein is complexed withan sgRNA that targets the complex to a specific target site in the DNA.The embodiment then involves the introduction of a trans prime editingRNA template (“tPERT”), which operates in place of a PEgRNA by providinga primer binding site (PBS) and an DNA synthesis template on separatemolecule, i.e., the tPERT, which is also equipped with a MS2 aptamer(stem loop). The MS2cp protein recruits the tPERT by binding to the MS2aptamer of the molecule. FIG. 72C depicts alternative designs forPEgRNAs that can be achieved through known methods for chemicalsynthesis of nucleic acid molecules. For example, chemical synthesis canbe used to synthesize a hybrid RNA/DNA PEgRNA molecule for use in primeediting, wherein the extension arm of the hybrid PEgRNA is DNA insteadof RNA. In such an embodiment, a DNA-dependent DNA polymerase can beused in place of a reverse transcriptase to synthesize the 3′ DNA flapcomprising the desired genetic change that is formed by prime editing.In another embodiment, the extension arm can be synthesized to include achemical linker that prevents the DNA polymerase (e.g., a reversetranscriptase) from using the sgRNA scaffold or backbone as a template.In still another embodiment, the extension arm may comprise a DNAsynthesis template that has the reverse orientation relative to theoverall orientation of the PEgRNA molecule. For example, and as shownfor a PEgRNA in the 5′-to-3′ orientation and with an extension attachedto the 3′ end of the sgRNA scaffold, the DNA synthesis template isorientated in the opposite direction, i.e., the 3′-to-5′ direction. Thisembodiment may be advantageous for PEgRNA embodiments with extensionarms positioned at the 3′ end of a gRNA. By reverse the orientation ofthe extension arm, the DNA synthesis by the polymerase (e.g., reversetranscriptase) will terminate once it reaches the newly orientated 5′ ofthe extension arm and will thus, not risk using the gRNA core as atemplate.

FIG. 73 demonstrates prime editing with tPERTs and the MS2 recruitmentsystem (aka MS2 tagging technique). An sgRNA targeting the prime editorprotein (PE2) to the target locus is expressed in combination with atPERT containing a primer binding site (a13-nt or 17-nt PBS), an RTtemplate encoding a His6 tag insertion and a homology arm, and an MS2aptamer (located at the 5′ or 3′ end of the tPERT molecule). Eitherprime editor protein (PE2) or a fusion of the MS2cp to the N-terminus ofPE2 was used. Editing was carried out with or without acomplementary-strand nicking sgRNA, as in the previously developed PE3system (designated in the x-axis as labels “PE2+nick” or “PE2”,respectively). This is also referred to and defined herein as“second-strand nicking.”

FIG. 74 demonstrates that the MS2 aptamer expression of the reversetranscriptase in trans and its recruitment with the MS2 aptamer system.The PEgRNAPEgRNA contains the MS2 RNA aptamer inserted into either oneof two sgRNA scaffold hairpins. The wild-type M-MLV reversetranscriptase is expressed as an N-terminal or C-terminal fusion to theMS2 coat protein (MCP). Editing is at the HEK3 site in HEK293T cells.

FIG. 75 provides a bar graph comparing the efficiency (i.e., “% of totalsequencing reads with the specified edit or indels”) of PE2, PE2-trunc,PE3, and PE3-trunc over different target sites in various cell lines.The data shows that the prime editors comprising the truncated RTvariants were about as efficient as the prime editors comprising thenon-truncated RT proteins.

FIG. 76 demonstrates the editing efficiency of intein-split primeeditors of Example 20. HEK239T cells were transfected with plasmidsencoding full-length PE2 or intein-split PE2, PEgRNA and nicking guideRNA. Consensus sequence (most amino-terminal residues of C terminalextein) are indicated. Percent editing at two sites in shown: HEK3+1 CTTinsertion and PRNP +6 G to T. Replicate n=3 independent transfections.See Example 20.

FIG. 77 demonstrates the editing efficiency of intein-split primeeditors of Example 20. Editing assessed by targeted deep sequencing inbulk cortex and GFP+ subpopulation upon delivery of 5E10vg per SpPE3half and a small amount 1E10 of nuclear-localized GFP:KASH to P0 mice byICV injection. Editors and GFP were packaged in AAV9 with EFS promoter.Mice were harvested three weeks post injection and GFP+ nuclei wereisolated by flow cytometry. Individual data points are shown, with 1-2mice per condition analyzed. See Example 20.

FIG. 78 demonstrates the editing efficiency of intein-split primeeditors of Example 20. Specifically, the figures depicts the AAVsplit-SpPE3 constructs using in Example 20. Co-transduction by AAVparticles separately expressing SpPE3-N and SpPE3-C recapitulates PE3activity. Note N-terminal genome contains a U6-sgRNA cassette expressingthe nicking sgRNA, and the C-terminal genome contains a U6-PEgRNAcassette expressing the PEgRNA. See Example 20.

FIG. 79 shows the editing efficiency of certain optimized linkers asdiscussed in Example 21. In particular, the data shows the editingefficiency of the PE2 construct with the current linker (noted asPE2—white box) compared to various versions with the linker replacedwith a sequence as indicated at the HEK3, EMX1, FANCF, RNF2 loci forrepresentative PEgRNAs for transition, transversion, insertion, anddeletion edits. The replacement linkers are referred to as “1×SGGS” (SEQID NO: 174), “2×SGGS” (SEQ ID NO: 446), “3×SGGS” (SEQ ID NO: 3889),“1×XTEN” (SEQ ID NO: 171), “no linker”, “lx Gly”, “1×Pro”, “1×EAAAK”(SEQ ID NO: 3968), “2×EAAAK”” (SEQ ID NO: 3969), and “3×EAAAK” (SEQ IDNO: 3970). The editing efficiency is measured in bar graph formatrelative to the “control” editing efficiency of PE2. The linker of PE2is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All editing wasdone in the context of the PE3 system, i.e., which refers the PE2editing construct plus the addition of the optimal secondary sgRNAnicking guide. See Example 21.

FIG. 80 . Taking the average fold efficacy relative to PE2 yields thegraph shown, indicating that use of a 1×XTEN (SEQ ID NO: 171) linkersequence improves editing efficiency by 1.14 fold on average (n=15). SeeExample 21.

FIG. 81 depicts the transcription level of PEgRNAs from differentpromoters, as described in Example 22.

FIG. 82 As depicted in Example 22, impact of different types ofmodifications on PEgRNA structure on editing efficiency relative tounmodified PEgRNA.

FIG. 83 Depicts a PE experiment that targeted editing of the HEK3 gene,specifically targeting the insertion of a 10 nt insertion at position +1relative to the nick site and using PE3. See Example 22.

FIG. 84A depicts an exemplary PEgRNA having a spacer, gRNA core, and anextension arm (RT template+primer binding site), which is modified atthe 3′ end of the PEgRNA with a tRNA molecule, coupled through a UCUlinker. The tRNA includes various post-transcriptional modifications.Said modification are not required, however.

FIG. 84B depicts structure of tRNA that can be used to modify PEgRNAstructures. See Example 22. The P1 can be variable in length. The P1 canbe extended to help prevent RNAseP processing of the PEgRNA-tRNA fusion.

FIG. 85 depicts a PE experiment that targeted editing of the FANCF gene,specifically targeting a G-to-T conversion at position +5 relative tothe nick site and using PE3 construct. See Example 22.

FIG. 86 depicts a PE experiment that targeted editing of the HEK3 gene,specifically targeting the insertion of a 71 nt FLAG tag insertion atposition +1 relative to the nick site and using PE3 construct. SeeExample 22.

FIG. 87 results from a screen in N2A cells where the pegRNA installs1412Adel, with details about the primer binding site (PBS) length andreverse transcriptase (RT) template length. (Shown with and withoutindels). See Example 23.

FIG. 88 results from a screen in N2A cells where the pegRNA installs1412Adel, with details about the primer binding site (PBS) length andreverse transcriptase (RT) template length. (Shown with and withoutindels). See Example 23.

FIG. 89 depicts results of editing at a proxy locus in the β-globin geneand at HEK3 in healthy HSCs, varying the concentration of editor topegRNA and nicking gRNA. See Example 23.

FIG. 90 is an exemplary schematic showing editing of a target sequenceby lineage PE guide #1 and #2. The sequences shown (top-bottom)correspond to SEQ ID NOs: 752, 752, 753, 754, 754, 755, and 756.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

Antisense Strand

In genetics, the “antisense” strand of a segment within double-strandedDNA is the template strand, and which is considered to run in the 3′ to5′ orientation. By contrast, the “sense” strand is the segment withindouble-stranded DNA that runs from 5′ to 3′, and which is complementaryto the antisense strand of DNA, or template strand, which runs from 3′to 5′. In the case of a DNA segment that encodes a protein, the sensestrand is the strand of DNA that has the same sequence as the mRNA,which takes the antisense strand as its template during transcription,and eventually undergoes (typically, not always) translation into aprotein. The antisense strand is thus responsible for the RNA that islater translated to protein, while the sense strand possesses a nearlyidentical makeup to that of the mRNA. Note that for each segment ofdsDNA, there will possibly be two sets of sense and antisense, dependingon which direction one reads (since sense and antisense is relative toperspective). It is ultimately the gene product, or mRNA, that dictateswhich strand of one segment of dsDNA is referred to as sense orantisense.

Bi-Specific Ligand

The term “bi-specific ligand” or “bi-specific moiety,” as used herein,refers to a ligand that binds to two different ligand-binding domains.In certain embodiments, the ligand is a small molecule compound, or apeptide, or a polypeptide. In other embodiments, ligand-binding domainis a “dimerization domain,” which can be install as a peptide tag onto aprotein. In various embodiments, two proteins each comprising the sameor different dimerization domains can be induced to dimerize through thebinding of each dimerization domain to the bi-specific ligand. As usedherein, “bi-specific ligands” may be equivalently refer to “chemicalinducers of dimerization” or “CIDs”.

Cas9

The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nucleasecomprising a Cas9 domain, or a fragment thereof (e.g., a proteincomprising an active or inactive DNA cleavage domain of Cas9, and/or thegRNA binding domain of Cas9). A “Cas9 domain” as used herein, is aprotein fragment comprising an active or inactive cleavage domain ofCas9 and/or the gRNA binding domain of Cas9. A “Cas9 protein” is a fulllength Cas9 protein. A Cas9 nuclease is also referred to sometimes as acasn1 nuclease or a CRISPR (Clustered Regularly Interspaced ShortPalindromic Repeat)-associated nuclease. CRISPR is an adaptive immunesystem that provides protection against mobile genetic elements(viruses, transposable elements, and conjugative plasmids). CRISPRclusters contain spacers, sequences complementary to antecedent mobileelements, and target invading nucleic acids. CRISPR clusters aretranscribed and processed into CRISPR RNA (crRNA). In type II CRISPRsystems correct processing of pre-crRNA requires a trans-encoded smallRNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 domain. ThetracrRNA serves as a guide for ribonuclease 3-aided processing ofpre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaveslinear or circular dsDNA target complementary to the spacer. The targetstrand not complementary to crRNA is first cut endonucleolytically, thentrimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavagetypically requires protein and both RNAs. However, single guide RNAs(“sgRNA”, or simply “gNRA”) can be engineered so as to incorporateaspects of both the crRNA and tracrRNA into a single RNA species. See,e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A.,Charpentier E. Science 337:816-821(2012), the entire contents of whichare hereby incorporated by reference. Cas9 recognizes a short motif inthe CRISPR repeat sequences (the PAM or protospacer adjacent motif) tohelp distinguish self versus non-self. Cas9 nuclease sequences andstructures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an M1 strain of Streptococcus pyogenes.”Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., SavicG., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H.S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L.,White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc.Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation bytrans-encoded small RNA and host factor RNase III.” Deltcheva E.,Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., EckertM. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “Aprogrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A.,Charpentier E. Science 337:816-821 (2012), the entire contents of eachof which are incorporated herein by reference). Cas9 orthologs have beendescribed in various species, including, but not limited to, S. pyogenesand S. thermophilus. Additional suitable Cas9 nucleases and sequenceswill be apparent to those of skill in the art based on this disclosure,and such Cas9 nucleases and sequences include Cas9 sequences from theorganisms and loci disclosed in Chylinski, Rhun, and Charpentier, “ThetracrRNA and Cas9 families of type II CRISPR-Cas immunity systems”(2013) RNA Biology 10:5, 726-737; the entire contents of which areincorporated herein by reference. In some embodiments, a Cas9 nucleasecomprises one or more mutations that partially impair or inactivate theDNA cleavage domain.

A nuclease-inactivated Cas9 domain may interchangeably be referred to asa “dCas9” protein (for nuclease-“dead” Cas9). Methods for generating aCas9 domain (or a fragment thereof) having an inactive DNA cleavagedomain are known (see, e.g., Jinek et al., Science. 337:816-821(2012);Qi et al., “Repurposing CRISPR as an RNA-Guided Platform forSequence-Specific Control of Gene Expression” (2013) Cell. 28;152(5):1173-83, the entire contents of each of which are incorporatedherein by reference). For example, the DNA cleavage domain of Cas9 isknown to include two subdomains, the HNH nuclease subdomain and theRuvC1 subdomain. The HNH subdomain cleaves the strand complementary tothe gRNA, whereas the RuvC1 subdomain cleaves the non-complementarystrand. Mutations within these subdomains can silence the nucleaseactivity of Cas9. For example, the mutations D10A and H840A completelyinactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al.,Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)).In some embodiments, proteins comprising fragments of Cas9 are provided.For example, in some embodiments, a protein comprises one of two Cas9domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavagedomain of Cas9. In some embodiments, proteins comprising Cas9 orfragments thereof are referred to as “Cas9 variants.” A Cas9 variantshares homology to Cas9, or a fragment thereof. For example, a Cas9variant is at least about 70% identical, at least about 80% identical,at least about 90% identical, at least about 95% identical, at leastabout 96% identical, at least about 97% identical, at least about 98%identical, at least about 99% identical, at least about 99.5% identical,at least about 99.8% identical, or at least about 99.9% identical towild type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). In some embodiments, theCas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, ormore amino acid changes compared to wild type Cas9 (e.g., SpCas9 of SEQID NO: 18). In some embodiments, the Cas9 variant comprises a fragmentof SEQ ID NO: 18 Cas9 (e.g., a gRNA binding domain or a DNA-cleavagedomain), such that the fragment is at least about 70% identical, atleast about 80% identical, at least about 90% identical, at least about95% identical, at least about 96% identical, at least about 97%identical, at least about 98% identical, at least about 99% identical,at least about 99.5% identical, or at least about 99.9% identical to thecorresponding fragment of wild type Cas9 (e.g., SpCas9 of SEQ ID NO:18). In some embodiments, the fragment is at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% identical, at least 96%, at least 97%, at least98%, at least 99%, or at least 99.5% of the amino acid length of acorresponding wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 18).

cDNA

The term “cDNA” refers to a strand of DNA copied from an RNA template.cDNA is complementary to the RNA template.

Circular Permutant

As used herein, the term “circular permutant” refers to a protein orpolypeptide (e.g., a Cas9) comprising a circular permutation, which ischange in the protein's structural configuration involving a change inorder of amino acids appearing in the protein's amino acid sequence. Inother words, circular permutants are proteins that have altered N- andC-termini as compared to a wild-type counterpart, e.g., the wild-typeC-terminal half of a protein becomes the new N-terminal half. Circularpermutation (or CP) is essentially the topological rearrangement of aprotein's primary sequence, connecting its N- and C-terminus, often witha peptide linker, while concurrently splitting its sequence at adifferent position to create new, adjacent N- and C-termini. The resultis a protein structure with different connectivity, but which often canhave the same overall similar three-dimensional (3D) shape, and possiblyinclude improved or altered characteristics, including, reducedproteolytic susceptibility, improved catalytic activity, alteredsubstrate or ligand binding, and/or improved thermostability. Circularpermutant proteins can occur in nature (e.g., concanavalin A andlectin). In addition, circular permutation can occur as a result ofposttranslational modifications or may be engineered using recombinanttechniques.

Circularly Permuted Cas9

The term “circularly permuted Cas9” refers to any Cas9 protein, orvariant thereof, that has been occurs as a circular permutant, wherebyits N- and C-termini have been topically rearranged. Such circularlypermuted Cas9 proteins (“CP-Cas9”), or variants thereof, retain theability to bind DNA when complexed with a guide RNA (gRNA). See, Oakeset al., “Protein Engineering of Cas9 for enhanced function,” MethodsEnzymol, 2014, 546: 491-511 and Oakes et al., “CRISPR-Cas9 CircularPermutants as Programmable Scaffolds for Genome Modification,” Cell,Jan. 10, 2019, 176: 254-267, each of are incorporated herein byreference. The instant disclosure contemplates any previously knownCP-Cas9 or use a new CP-Cas9 so long as the resulting circularlypermuted protein retains the ability to bind DNA when complexed with aguide RNA (gRNA). Exemplary CP-Cas9 proteins are SEQ ID NOs: 77-86.

CRISPR

CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteriaand archaea that represent snippets of prior infections by a virus thathave invaded the prokaryote. The snippets of DNA are used by theprokaryotic cell to detect and destroy DNA from subsequent attacks bysimilar viruses and effectively compose, along with an array ofCRISPR-associated proteins (including Cas9 and homologs thereof) andCRISPR-associated RNA, a prokaryotic immune defense system. In nature,CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).In certain types of CRISPR systems (e.g., type II CRISPR systems),correct processing of pre-crRNA requires a trans-encoded small RNA(tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. ThetracrRNA serves as a guide for ribonuclease 3-aided processing ofpre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaveslinear or circular dsDNA target complementary to the RNA. Specifically,the target strand not complementary to crRNA is first cutendonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature,DNA-binding and cleavage typically requires protein and both RNAs.However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineeredso as to incorporate aspects of both the crRNA and tracrRNA into asingle RNA species—the guide RNA. See, e.g., Jinek M., Chylinski K.,Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science337:816-821(2012), the entire contents of which is hereby incorporatedby reference. Cas9 recognizes a short motif in the CRISPR repeatsequences (the PAM or protospacer adjacent motif) to help distinguishself versus non-self. CRISPR biology, as well as Cas9 nuclease sequencesand structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an M1 strain of Streptococcus pyogenes.”Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., SavicG., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H.S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L.,White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc.Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation bytrans-encoded small RNA and host factor RNase III.” Deltcheva E.,Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., EckertM. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “Aprogrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A.,Charpentier E. Science 337:816-821(2012), the entire contents of each ofwhich are incorporated herein by reference). Cas9 orthologs have beendescribed in various species, including, but not limited to, S. pyogenesand S. thermophilus. Additional suitable Cas9 nucleases and sequenceswill be apparent to those of skill in the art based on this disclosure,and such Cas9 nucleases and sequences include Cas9 sequences from theorganisms and loci disclosed in Chylinski, Rhun, and Charpentier, “ThetracrRNA and Cas9 families of type II CRISPR-Cas immunity systems”(2013) RNA Biology 10:5, 726-737; the entire contents of which areincorporated herein by reference.

In certain types of CRISPR systems (e.g., type II CRISPR systems),correct processing of pre-crRNA requires a trans-encoded small RNA(tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 protein. ThetracrRNA serves as a guide for ribonuclease 3-aided processing ofpre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaveslinear or circular nucleic acid target complementary to the RNA.Specifically, the target strand not complementary to crRNA is first cutendonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature,DNA-binding and cleavage typically requires protein and both RNAs.However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineeredso as to incorporate embodiments of both the crRNA and tracrRNA into asingle RNA species—the guide RNA.

In general, a “CRISPR system” refers collectively to transcripts andother elements involved in the expression of or directing the activityof CRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or anactive partial tracrRNA), a tracr mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus. The tracrRNA of thesystem is complementary (fully or partially) to the tracr mate sequencepresent on the guide RNA.

DNA Synthesis Template

As used herein, the term “DNA synthesis template” refers to the regionor portion of the extension arm of a PEgRNA that is utilized as atemplate strand by a polymerase of a prime editor to encode a 3′single-strand DNA flap that contains the desired edit and which then,through the mechanism of prime editing, replaces the correspondingendogenous strand of DNA at the target site. In various embodiments, theDNA synthesis template is shown in FIG. 3A (in the context of a PEgRNAcomprising a 5′ extension arm), FIG. 3B (in the context of a PEgRNAcomprising a 3′ extension arm), FIG. 3C (in the context of an internalextension arm), FIG. 3D (in the context of a 3′ extension arm), and FIG.3E (in the context of a 5′ extension arm). The extension arm, includingthe DNA synthesis template, may be comprised of DNA or RNA. In the caseof RNA, the polymerase of the prime editor can be an RNA-dependent DNApolymerase (e.g., a reverse transcriptase). In the case of DNA, thepolymerase of the prime editor can be a DNA-dependent DNA polymerase. Invarious embodiments (e.g., as depicted in FIGS. 3D-3E), the DNAsynthesis template (4) may comprise the “edit template” and the“homology arm”, and all or a portion of the optional 5′ end modifierregion, e2. That is, depending on the nature of the e2 region (e.g.,whether it includes a hairpin, toeloop, or stem/loop secondarystructure), the polymerase may encode none, some, or all of the e2region, as well. Said another way, in the case of a 3′ extension arm,the DNA synthesis template (3) can include the portion of the extensionarm (3) that spans from the 5′ end of the primer binding site (PBS) to3′ end of the gRNA core that may operate as a template for the synthesisof a single-strand of DNA by a polymerase (e.g., a reversetranscriptase). In the case of a 5′ extension arm, the DNA synthesistemplate (3) can include the portion of the extension arm (3) that spansfrom the 5′ end of the PEgRNA molecule to the 3′ end of the edittemplate. Preferably, the DNA synthesis template excludes the primerbinding site (PBS) of PEgRNAs either having a 3′ extension arm or a 5′extension arm. Certain embodiments described here (e.g, FIG. 71A) referto an “an RT template,” which is inclusive of the edit template and thehomology arm, i.e., the sequence of the PEgRNA extension arm which isactually used as a template during DNA synthesis. The term “RT template”is equivalent to the term “DNA synthesis template.”

In the case of trans prime editing (e.g., FIG. 3G and FIG. 3H), theprimer binding site (PBS) and the DNA synthesis template can beengineered into a separate molecule referred to as a trans prime editorRNA template (tPERT).

Dimerization Domain

The term “dimerization domain” refers to a ligand-binding domain thatbinds to a binding moiety of a bi-specific ligand. A “first”dimerization domain binds to a first binding moiety of a bi-specificligand and a “second” dimerization domain binds to a second bindingmoiety of the same bi-specific ligand. When the first dimerizationdomain is fused to a first protein (e.g., via PE, as discussed herein)and the second dimerization domain (e.g., via PE, as discussed herein)is fused to a second protein, the first and second protein dimerize inthe presence of a bi-specific ligand, wherein the bi-specific ligand hasat least one moiety that binds to the first dimerization domain and atleast another moiety that binds to the second dimerization domain.

Downstream

As used herein, the terms “upstream” and “downstream” are terms ofrelativity that define the linear position of at least two elementslocated in a nucleic acid molecule (whether single or double-stranded)that is orientated in a 5′-to-3′ direction. In particular, a firstelement is upstream of a second element in a nucleic acid molecule wherethe first element is positioned somewhere that is 5′ to the secondelement. For example, a SNP is upstream of a Cas9-induced nick site ifthe SNP is on the 5′ side of the nick site. Conversely, a first elementis downstream of a second element in a nucleic acid molecule where thefirst element is positioned somewhere that is 3′ to the second element.For example, a SNP is downstream of a Cas9-induced nick site if the SNPis on the 3′ side of the nick site. The nucleic acid molecule can be aDNA (double or single stranded). RNA (double or single stranded), or ahybrid of DNA and RNA. The analysis is the same for single strandnucleic acid molecule and a double strand molecule since the termsupstream and downstream are in reference to only a single strand of anucleic acid molecule, except that one needs to select which strand ofthe double stranded molecule is being considered. Often, the strand of adouble stranded DNA which can be used to determine the positionalrelativity of at least two elements is the “sense” or “coding” strand.In genetics, a “sense” strand is the segment within double-stranded DNAthat runs from 5′ to 3′, and which is complementary to the antisensestrand of DNA, or template strand, which runs from 3′ to 5′. Thus, as anexample, a SNP nucleobase is “downstream” of a promoter sequence in agenomic DNA (which is double-stranded) if the SNP nucleobase is on the3′ side of the promoter on the sense or coding strand.

Edit Template

The term “edit template” refers to a portion of the extension arm thatencodes the desired edit in the single strand 3′ DNA flap that issynthesized by the polymerase, e.g., a DNA-dependent DNA polymerase,RNA-dependent DNA polymerase (e.g., a reverse transcriptase). Certainembodiments described here (e.g., FIG. 71A) refer to “an RT template,”which refers to both the edit template and the homology arm together,i.e., the sequence of the PEgRNA extension arm which is actually used asa template during DNA synthesis. The term “RT edit template” is alsoequivalent to the term “DNA synthesis template,” but wherein the RT edittemplate reflects the use of a prime editor having a polymerase that isa reverse transcriptase, and wherein the DNA synthesis template reflectsmore broadly the use of a prime editor having any polymerase.

Effective Amount

The term “effective amount,” as used herein, refers to an amount of abiologically active agent that is sufficient to elicit a desiredbiological response. For example, in some embodiments, an effectiveamount of a prime editor (PE) may refer to the amount of the editor thatis sufficient to edit a target site nucleotide sequence, e.g., a genome.In some embodiments, an effective amount of a prime editor (PE) providedherein, e.g., of a fusion protein comprising a nickase Cas9 domain and areverse transcriptase may refer to the amount of the fusion protein thatis sufficient to induce editing of a target site specifically bound andedited by the fusion protein. As will be appreciated by the skilledartisan, the effective amount of an agent, e.g., a fusion protein, anuclease, a hybrid protein, a protein dimer, a complex of a protein (orprotein dimer) and a polynucleotide, or a polynucleotide, may varydepending on various factors as, for example, on the desired biologicalresponse, e.g., on the specific allele, genome, or target site to beedited, on the cell or tissue being targeted, and on the agent beingused.

Error-Prone Reverse Transcriptase

As used herein, the term “error-prone” reverse transcriptase (or morebroadly, any polymerase) refers to a reverse transcriptase (or morebroadly, any polymerase) that occurs naturally or which has been derivedfrom another reverse transcriptase (e.g., a wild type M-MLV reversetranscriptase) which has an error rate that is less than the error rateof wild type M-MLV reverse transcriptase. The error rate of wild typeM-MLV reverse transcriptase is reported to be in the range of one errorin 15,000 (higher) to 27,000 (lower). An error rate of 1 in 15,000corresponds with an error rate of 6.7×10⁻⁵. An error rate of 1 in 27,000corresponds with an error rate of 3.7×10⁻⁵. See Boutabout et al. (2001)“DNA synthesis fidelity by the reverse transcriptase of the yeastretrotransposon Ty1,” Nucleic Acids Res 29(11):2217-2222, which isincorporated herein by reference. Thus, for purposes of thisapplication, the term “error prone” refers to those RT that have anerror rate that is greater than one error in 15,000 nucleobaseincorporation (6.7×10⁻⁵ or higher), e.g., 1 error in 14,000 nucleobases(7.14×10⁻⁵ or higher), 1 error in 13,000 nucleobases or fewer (7.7×10⁻⁵or higher), 1 error in 12,000 nucleobases or fewer (7.7×10⁻⁵ or higher),1 error in 11,000 nucleobases or fewer (9.1×10⁻⁵ or higher), 1 error in10,000 nucleobases or fewer (1×10⁻⁴ or 0.0001 or higher), 1 error in9,000 nucleobases or fewer (0.00011 or higher), 1 error in 8,000nucleobases or fewer (0.00013 or higher) 1 error in 7,000 nucleobases orfewer (0.00014 or higher), 1 error in 6,000 nucleobases or fewer(0.00016 or higher), 1 error in 5,000 nucleobases or fewer (0.0002 orhigher), 1 error in 4,000 nucleobases or fewer (0.00025 or higher), 1error in 3,000 nucleobases or fewer (0.00033 or higher), 1 error in2,000 nucleobase or fewer (0.00050 or higher), or 1 error in 1,000nucleobases or fewer (0.001 or higher), or 1 error in 500 nucleobases orfewer (0.002 or higher), or 1 error in 250 nucleobases or fewer (0.004or higher).

Extein

The term “extein,” as used herein, refers to an polypeptide sequencethat is flanked by an intein and is ligated to another extein during theprocess of protein splicing to form a mature, spliced protein.Typically, an intein is flanked by two extein sequences that are ligatedtogether when the intein catalyzes its own excision. Exteins,accordingly, are the protein analog to exons found in mRNA. For example,a polypeptide comprising an intein may be of the structureextein(N)-intein-extein(C). After excision of the intein and splicing ofthe two exteins, the resulting structures are extein(N)-extein(C) and afree intein. In various configurations, the exteins may be separateproteins (e.g., half of a Cas9 or PE fusion protein), each fused to asplit-intein, wherein the excision of the split inteins causes thesplicing together of the extein sequences.

Extension Arm

The term “extension arm” refers to a nucleotide sequence component of aPEgRNA which provides several functions, including a primer binding siteand an edit template for reverse transcriptase. In some embodiments,e.g., FIG. 3D, the extension arm is located at the 3′ end of the guideRNA. In other embodiments, e.g., FIG. 3E, the extension arm is locatedat the 5′ end of the guide RNA. In some embodiments, the extension armalso includes a homology arm. In various embodiments, the extension armcomprises the following components in a 5′ to 3′ direction: the homologyarm, the edit template, and the primer binding site. Sincepolymerization activity of the reverse transcriptase is in the 5′ to 3′direction, the preferred arrangement of the homology arm, edit template,and primer binding site is in the 5′ to 3′ direction such that thereverse transcriptase, once primed by an annealed primer sequence,polymerases a single strand of DNA using the edit template as acomplementary template strand. Further details, such as the length ofthe extension arm, are described elsewhere herein.

The extension arm may also be described as comprising generally tworegions: a primer binding site (PBS) and a DNA synthesis template, asshown in FIG. 3G (top), for instance. The primer binding site binds tothe primer sequence that is formed from the endogenous DNA strand of thetarget site when it becomes nicked by the prime editor complex, therebyexposing a 3′ end on the endogenous nicked strand. As explained herein,the binding of the primer sequence to the primer binding site on theextension arm of the PEgRNA creates a duplex region with an exposed 3′end (i.e., the 3′ of the primer sequence), which then provides asubstrate for a polymerase to begin polymerizing a single strand of DNAfrom the exposed 3′ end along the length of the DNA synthesis template.The sequence of the single strand DNA product is the complement of theDNA synthesis template. Polymerization continues towards the 5′ of theDNA synthesis template (or extension arm) until polymerizationterminates. Thus, the DNA synthesis template represents the portion ofthe extension arm that is encoded into a single strand DNA product(i.e., the 3′ single strand DNA flap containing the desired genetic editinformation) by the polymerase of the prime editor complex and whichultimately replaces the corresponding endogenous DNA strand of thetarget site that sits immediate downstream of the PE-induced nick site.Without being bound by theory, polymerization of the DNA synthesistemplate continues towards the 5′ end of the extension arm until atermination event. Polymerization may terminate in a variety of ways,including, but not limited to (a) reaching a 5′ terminus of the PEgRNA(e.g., in the case of the 5′ extension arm wherein the DNA polymerasesimply runs out of template), (b) reaching an impassable RNA secondarystructure (e.g., hairpin or stem/loop), or (c) reaching a replicationtermination signal, e.g., a specific nucleotide sequence that blocks orinhibits the polymerase, or a nucleic acid topological signal, such as,supercoiled DNA or RNA.

Flap Endonuclease (e.g., FEN1)

As used herein, the term “flap endonuclease” refers to an enzyme thatcatalyzes the removal of 5′ single strand DNA flaps. These are naturallyoccurring enzymes that process the removal of 5′ flaps formed duringcellular processes, including DNA replication. The prime editing methodsherein described may utilize endogenously supplied flap endonucleases orthose provided in trans to remove the 5′ flap of endogenous DNA formedat the target site during prime editing. Flap endonucleases are known inthe art and can be found described in Patel et al., “Flap endonucleasespass 5′-flaps through a flexible arch using a disorder-thread-ordermechanism to confer specificity for free 5′-ends,” Nucleic AcidsResearch, 2012, 40(10): 4507-4519, Tsutakawa et al., “Human flapendonuclease structures, DNA double-base flipping, and a unifiedunderstanding of the FEN1 superfamily,” Cell, 2011, 145(2): 198-211, andBalakrishnan et al., “Flap Endonuclease 1,” Annu Rev Biochem, 2013, Vol82: 119-138 (each of which are incorporated herein by reference). Anexemplary flap endonuclease is FEN1, which can be represented by thefollowing amino acid sequence:

DESCRIP- SEQ ID  TION SEQUENCE NO: FEN1MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAI SEQ ID WILDDASMSIYQFLIAVRQGGDVLQNEEGETTSHLMG NO: 7 TYPEMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAK RSERRAEAEKQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAA LVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLG SDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELK WSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEPKGS TKKKAKTGAAGKFKRGK

Functional Equivalent

The term “functional equivalent” refers to a second biomolecule that isequivalent in function, but not necessarily equivalent in structure to afirst biomolecule. For example, a “Cas9 equivalent” refers to a proteinthat has the same or substantially the same functions as Cas9, but notnecessarily the same amino acid sequence. In the context of thedisclosure, the specification refers throughout to “a protein X, or afunctional equivalent thereof.” In this context, a “functionalequivalent” of protein X embraces any homolog, paralog, fragment,naturally occurring, engineered, mutated, or synthetic version ofprotein X which bears an equivalent function.

Fusion Protein

The term “fusion protein” as used herein refers to a hybrid polypeptidewhich comprises protein domains from at least two different proteins.One protein may be located at the amino-terminal (N-terminal) portion ofthe fusion protein or at the carboxy-terminal (C-terminal) protein thusforming an “amino-terminal fusion protein” or a “carboxy-terminal fusionprotein,” respectively. A protein may comprise different domains, forexample, a nucleic acid binding domain (e.g., the gRNA binding domain ofCas9 that directs the binding of the protein to a target site) and anucleic acid cleavage domain or a catalytic domain of a nucleic-acidediting protein. Another example includes a Cas9 or equivalent thereofto a reverse transcriptase. Any of the proteins provided herein may beproduced by any method known in the art. For example, the proteinsprovided herein may be produced via recombinant protein expression andpurification, which is especially suited for fusion proteins comprisinga peptide linker. Methods for recombinant protein expression andpurification are well known, and include those described by Green andSambrook, Molecular Cloning: A Laboratory Manual (4^(th) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), theentire contents of which are incorporated herein by reference.

Gene of Interest (GOI)

The term “gene of interest” or “GOI” refers to a gene that encodes abiomolecule of interest (e.g., a protein or an RNA molecule). A proteinof interest can include any intracellular protein, membrane protein, orextracellular protein, e.g., a nuclear protein, transcription factor,nuclear membrane transporter, intracellular organelle associatedprotein, a membrane receptor, a catalytic protein, and enzyme, atherapeutic protein, a membrane protein, a membrane transport protein, asignal transduction protein, or an immunological protein (e.g., an IgGor other antibody protein), etc. The gene of interest may also encode anRNA molecule, including, but not limited to, messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA),antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA(siRNA), and cell-free RNA (cfRNA).

Guide RNA (“gRNA”)

As used herein, the term “guide RNA” is a particular type of guidenucleic acid which is mostly commonly associated with a Cas protein of aCRISPR-Cas9 and which associates with Cas9, directing the Cas9 proteinto a specific sequence in a DNA molecule that includes complementarityto protospacer sequence of the guide RNA. However, this term alsoembraces the equivalent guide nucleic acid molecules that associate withCas9 equivalents, homologs, orthologs, or paralogs, whether naturallyoccurring or non-naturally occurring (e.g., engineered or recombinant),and which otherwise program the Cas9 equivalent to localize to aspecific target nucleotide sequence. The Cas9 equivalents may includeother napDNAbp from any type of CRISPR system (e.g., type II, V, VI),including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cassystem), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type VCRISPR-Cas system). Further Cas-equivalents are described in Makarova etal., “C2c2 is a single-component programmable RNA-guided RNA-targetingCRISPR effector,” Science 2016; 353(6299), the contents of which areincorporated herein by reference. Exemplary sequences are and structuresof guide RNAs are provided herein. In addition, methods for designingappropriate guide RNA sequences are provided herein. As used herein, the“guide RNA” may also be referred to as a “traditional guide RNA” tocontrast it with the modified forms of guide RNA termed “prime editingguide RNAs” (or “PEgRNAs”) which have been invented for the primeediting methods and composition disclosed herein.

Guide RNAs or PEgRNAs may comprise various structural elements thatinclude, but are not limited to:

Spacer sequence—the sequence in the guide RNA or PEgRNA (having about 20nts in length) which binds to the protospacer in the target DNA.

gRNA core (or gRNA scaffold or backbone sequence)—refers to the sequencewithin the gRNA that is responsible for Cas9 binding, it does notinclude the 20 bp spacer/targeting sequence that is used to guide Cas9to target DNA.

Extension arm—a single strand extension at the 3′ end or the 5′ end ofthe PEgRNA which comprises a primer binding site and a DNA synthesistemplate sequence that encodes via a polymerase (e.g., a reversetranscriptase) a single stranded DNA flap containing the genetic changeof interest, which then integrates into the endogenous DNA by replacingthe corresponding endogenous strand, thereby installing the desiredgenetic change.

Transcription terminator—the guide RNA or PEgRNA may comprise atranscriptional termination sequence at the 3′ of the molecule.

Homology Arm

The term “homology arm” refers to a portion of the extension arm thatencodes a portion of the resulting reverse transcriptase-encoded singlestrand DNA flap that is to be integrated into the target DNA site byreplacing the endogenous strand. The portion of the single strand DNAflap encoded by the homology arm is complementary to the non-editedstrand of the target DNA sequence, which facilitates the displacement ofthe endogenous strand and annealing of the single strand DNA flap in itsplace, thereby installing the edit. This component is further definedelsewhere. The homology arm is part of the DNA synthesis template sinceit is by definition encoded by the polymerase of the prime editorsdescribed herein.

Host Cell

The term “host cell,” as used herein, refers to a cell that can host,replicate, and express a vector described herein, e.g., a vectorcomprising a nucleic acid molecule encoding a fusion protein comprisinga Cas9 or Cas9 equivalent and a reverse transcriptase.

Inteins

As used herein, the term “intein” refers to auto-processing polypeptidedomains found in organisms from all domains of life. An intein(intervening protein) carries out a unique auto-processing event knownas protein splicing in which it excises itself out from a largerprecursor polypeptide through the cleavage of two peptide bonds and, inthe process, ligates the flanking extein (external protein) sequencesthrough the formation of a new peptide bond. This rearrangement occurspost-translationally (or possibly co-translationally), as intein genesare found embedded in frame within other protein-coding genes.Furthermore, intein-mediated protein splicing is spontaneous; itrequires no external factor or energy source, only the folding of theintein domain. This process is also known as cis-protein splicing, asopposed to the natural process of trans-protein splicing with “splitinteins.” Inteins are the protein equivalent of the self-splicing RNAintrons (see Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)),which catalyze their own excision from a precursor protein with theconcomitant fusion of the flanking protein sequences, known as exteins(reviewed in Perler et al., Curr. Opin. Chem. Biol. 1:292-299 (1997);Perler, F. B. Cell 92(1):1-4 (1998); Xu et al., EMBO J. 15(19):5146-5153(1996)).

As used herein, the term “protein splicing” refers to a process in whichan interior region of a precursor protein (an intein) is excised and theflanking regions of the protein (exteins) are ligated to form the matureprotein. This natural process has been observed in numerous proteinsfrom both prokaryotes and eukaryotes (Perler, F. B., Xu, M. Q., Paulus,H. Current Opinion in Chemical Biology 1997, 1, 292-299; Perler, F. B.Nucleic Acids Research 1999, 27, 346-347). The intein unit contains thenecessary components needed to catalyze protein splicing and oftencontains an endonuclease domain that participates in intein mobility(Perler, F. B., Davis, E. O., Dean, G. E., Gimble, F. S., Jack, W. E.,Neff, N., Noren, C. J., Thomer, J., Belfort, M. Nucleic Acids Research1994, 22, 1127-1127). The resulting proteins are linked, however, notexpressed as separate proteins. Protein splicing may also be conductedin trans with split inteins expressed on separate polypeptidesspontaneously combine to form a single intein which then undergoes theprotein splicing process to join to separate proteins.

The elucidation of the mechanism of protein splicing has led to a numberof intein-based applications (Comb, et al., U.S. Pat. No. 5,496,714;Comb, et al., U.S. Pat. No. 5,834,247; Camarero and Muir, J. Amer. Chem.Soc., 121:5597-5598 (1999); Chong, et al., Gene, 192:271-281 (1997),Chong, et al., Nucleic Acids Res., 26:5109-5115 (1998); Chong, et al.,J. Biol. Chem., 273:10567-10577 (1998); Cotton, et al. J. Am. Chem.Soc., 121:1100-1101 (1999); Evans, et al., J. Biol. Chem.,274:18359-18363 (1999); Evans, et al., J. Biol. Chem., 274:3923-3926(1999); Evans, et al., Protein Sci., 7:2256-2264 (1998); Evans, et al.,J. Biol. Chem., 275:9091-9094 (2000); Iwai and Pluckthun, FEBS Lett.459:166-172 (1999); Mathys, et al., Gene, 231:1-13 (1999); Mills, etal., Proc. Natl. Acad. Sci. USA 95:3543-3548 (1998); Muir, et al., Proc.Natl. Acad. Sci. USA 95:6705-6710 (1998); Otomo, et al., Biochemistry38:16040-16044 (1999); Otomo, et al., J. Biolmol. NMR 14:105-114 (1999);Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999);Severinov and Muir, J. Biol. Chem., 273:16205-16209 (1998);Shingledecker, et al., Gene, 207:187-195 (1998); Southworth, et al.,EMBO J. 17:918-926 (1998); Southworth, et al., Biotechniques, 27:110-120(1999); Wood, et al., Nat. Biotechnol., 17:889-892 (1999); Wu, et al.,Proc. Natl. Acad. Sci. USA 95:9226-9231 (1998a); Wu, et al., BiochimBiophys Acta 1387:422-432 (1998b); Xu, et al., Proc. Natl. Acad. Sci.USA 96:388-393 (1999); Yamazaki, et al., J. Am. Chem. Soc.,120:5591-5592 (1998)). Each reference is incorporated herein byreference.

Ligand-Dependent Intein

The term “ligand-dependent intein,” as used herein refers to an inteinthat comprises a ligand-binding domain. Typically, the ligand-bindingdomain is inserted into the amino acid sequence of the intein, resultingin a structure intein (N)-ligand-binding domain-intein (C). Typically,ligand-dependent inteins exhibit no or only minimal protein splicingactivity in the absence of an appropriate ligand, and a marked increaseof protein splicing activity in the presence of the ligand. In someembodiments, the ligand-dependent intein does not exhibit observablesplicing activity in the absence of ligand but does exhibit splicingactivity in the presence of the ligand. In some embodiments, theligand-dependent intein exhibits an observable protein splicing activityin the absence of the ligand, and a protein splicing activity in thepresence of an appropriate ligand that is at least 5 times, at least 10times, at least 50 times, at least 100 times, at least 150 times, atleast 200 times, at least 250 times, at least 500 times, at least 1000times, at least 1500 times, at least 2000 times, at least 2500 times, atleast 5000 times, at least 10000 times, at least 20000 times, at least25000 times, at least 50000 times, at least 100000 times, at least500000 times, or at least 1000000 times greater than the activityobserved in the absence of the ligand. In some embodiments, the increasein activity is dose dependent over at least 1 order of magnitude, atleast 2 orders of magnitude, at least 3 orders of magnitude, at least 4orders of magnitude, or at least 5 orders of magnitude, allowing forfine-tuning of intein activity by adjusting the concentration of theligand. Suitable ligand-dependent inteins are known in the art, and ininclude those provided below and those described in published U.S.Patent Application U.S. 2014/0065711 A1; Mootz et al., “Protein splicingtriggered by a small molecule.” J. Am. Chem. Soc. 2002; 124, 9044-9045;Mootz et al., “Conditional protein splicing: a new tool to controlprotein structure and function in vitro and in vivo.” J. Am. Chem. Soc.2003; 125, 10561-10569; Buskirk et al., Proc. Natl. Acad. Sci. USA.2004; 101, 10505-10510); Skretas & Wood, “Regulation of protein activitywith small-molecule-controlled inteins.” Protein Sci. 2005; 14, 523-532;Schwartz, et al., “Post-translational enzyme activation in an animal viaoptimized conditional protein splicing.” Nat. Chem. Biol. 2007; 3,50-54; Peck et al., Chem. Biol. 2011; 18 (5), 619-630; the entirecontents of each are hereby incorporated by reference. Exemplarysequences are as follows:

NAME SEQUENCE OF LIGAND-DEPENDENT INTEIN 2-4CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLL INTEIN:ARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HNC (SEQ ID NO: 8) 3-2CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLL INTEINARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYTNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HNC (SEQ ID NO: 9) 30R3-1CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLL INTEINARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HNC (SEQ ID NO: 10) 30R3-2CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLL INTEINARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HNC (SEQ ID NO: 11) 30R3-3CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLL INTEINARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HNC (SEQ ID NO: 12) 37R3-1CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLL INTEINARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYNPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HNC ((SEQ ID NO: 13) 37R3-2CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLL INTEINARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HNC (SEQ ID NO: 14) 37R3-3CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLL INTEINARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVV HNC (SEQ ID NO: 15)

Linker

The term “linker,” as used herein, refers to a molecule linking twoother molecules or moieties. The linker can be an amino acid sequence inthe case of a linker joining two fusion proteins. For example, a Cas9can be fused to a reverse transcriptase by an amino acid linkersequence. The linker can also be a nucleotide sequence in the case ofjoining two nucleotide sequences together. For example, in the instantcase, the traditional guide RNA is linked via a spacer or linkernucleotide sequence to the RNA extension of a prime editing guide RNAwhich may comprise a RT template sequence and an RT primer binding site.In other embodiments, the linker is an organic molecule, group, polymer,or chemical moiety. In some embodiments, the linker is 5-100 amino acidsin length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45,45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 aminoacids in length. Longer or shorter linkers are also contemplated.

Isolated

“Isolated” means altered or removed from the natural state. For example,a nucleic 20 acid or a peptide naturally present in a living animal isnot “isolated,” but the same nucleic acid or peptide partially orcompletely separated from the coexisting materials of its natural stateis “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In some embodiments, a gene of interest is encoded by an isolatednucleic acid. As used herein, the term “isolated,” refers to thecharacteristic of a material as provided herein being removed from itsoriginal or native environment (e.g., the natural environment if it isnaturally occurring). Therefore, a naturally-occurring polynucleotide orprotein or polypeptide present in a living animal is not isolated, butthe same polynucleotide or polypeptide, separated by human interventionfrom some or all of the coexisting materials in the natural system, isisolated. An artificial or engineered material, for example, anon-naturally occurring nucleic acid construct, such as the expressionconstructs and vectors described herein, are, accordingly, also referredto as isolated. A material does not have to be purified in order to beisolated. Accordingly, a material may be part of a vector and/or part ofa composition, and still be isolated in that such vector or compositionis not part of the environment in which the material is found in nature.

MS2 Tagging Technique

In various embodiments (e.g., as depicted in the embodiments of FIGS.72-73 and in Example 19), the term “MS2 tagging technique” refers to thecombination of an “RNA-protein interaction domain” (aka “RNA-proteinrecruitment domain or protein”) paired up with an RNA-binding proteinthat specifically recognizes and binds to the RNA-protein interactiondomain, e.g., a specific hairpin structure. These types of systems canbe leveraged to recruit a variety of functionalities to a prime editorcomplex that is bound to a target site. The MS2 tagging technique isbased on the natural interaction of the MS2 bacteriophage coat protein(“MCP” or “MS2cp”) with a stem-loop or hairpin structure present in thegenome of the phage, i.e., the “MS2 hairpin.” In the case of primeediting, the MS2 tagging technique comprises introducing the MS2 hairpininto a desired RNA molecule involved in prime editing (e.g., a PEgRNA ora tPERT), which then constitutes a specific interactable binding targetfor an RNA-binding protein that recognizes and binds to that structure.In the case of the MS2 hairpin, it is recognized and bound by the MS2bacteriophage coat protein (MCP). And, if MCP is fused to anotherprotein (e.g., a reverse transcriptase or other DNA polymerase), thenthe MS2 hairpin may be used to “recruit” that other protein in trans tothe target site occupied by the prime editing complex.

The prime editors described herein may incorporate as an aspect anyknown RNA-protein interaction domain to recruit or “co-localize”specific functions of interest to a prime editor complex. A review ofother modular RNA-protein interaction domains are described in the art,for example, in Johansson et al., “RNA recognition by the MS2 phage coatprotein,” Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al.,“Organization of intracellular reactions with rationally designed RNAassemblies,” Science, 2011, Vol. 333: 470-474; Mali et al., “Cas9transcriptional activators for target specificity screening and pairednickases for cooperative genome engineering,” Nat. Biotechnol., 2013,Vol. 31: 833-838; and Zalatan et al., “Engineering complex synthetictranscriptional programs with CRISPR RNA scaffolds,” Cell, 2015, Vol.160: 339-350, each of which are incorporated herein by reference intheir entireties. Other systems include the PP7 hairpin, whichspecifically recruits the PCP protein, and the “com” hairpin, whichspecifically recruits the Com protein. See Zalatan et al.

The nucleotide sequence of the MS2 hairpin (or equivalently referred toas the “MS2 aptamer”) is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO:763).

The amino acid sequence of the MCP or MS2cp is:

(SEQ ID NO: 764) GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY.

The MS2 hairpin (or “MS2 aptamer”) may also be referred to as a type of“RNA effector recruitment domain” (or equivalently as “RNA-bindingprotein recruitment domain” or simply as “recruitment domain”) since itis a physical structure (e.g., a hairpin) that is installed into aPEgRNA or tPERT that effectively recruits other effector functions(e.g., RNA-binding proteins having various functions, such as DNApolymerases or other DNA-modifying enzymes) to the PEgRNA or rPERT thatis so modified, and thus, co-localizing effector functions in trans tothe prime editing machinery. This application is not intended to belimited in any way to any particular RNA effector recruitment domainsand may include any available such domain, including the MS2 hairpin.Example 19 and FIG. 72(b) depicts the use of the MS2 aptamer joined to aDNA synthesis domain (i.e., the tPERT molecule) and a prime editor thatcomprises an MS2cp protein fused to a PE2 to cause the co-localizationof the prime editor complex (MS2cp-PE2:sgRNA complex) bound to thetarget DNA site and the DNA synthesis domain of the tPERT molecule.

napDNAbp

As used herein, the term “nucleic acid programmable DNA binding protein”or “napDNAbp,” of which Cas9 is an example, refer to a proteins whichuse RNA:DNA hybridization to target and bind to specific sequences in aDNA molecule. Each napDNAbp is associated with at least one guidenucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNAsequence that comprises a DNA strand (i.e., a target strand) that iscomplementary to the guide nucleic acid, or a portion thereof (e.g., theprotospacer of a guide RNA). In other words, the guide nucleic-acid“programs” the napDNAbp (e.g., Cas9 or equivalent) to localize and bindto a complementary sequence.

Without being bound by theory, the binding mechanism of a napDNAbp-guideRNA complex, in general, includes the step of forming an R-loop wherebythe napDNAbp induces the unwinding of a double-strand DNA target,thereby separating the strands in the region bound by the napDNAbp. Theguide RNA protospacer then hybridizes to the “target strand.” Thisdisplaces a “non-target strand” that is complementary to the targetstrand, which forms the single strand region of the R-loop. In someembodiments, the napDNAbp includes one or more nuclease activities,which then cut the DNA leaving various types of lesions. For example,the napDNAbp may comprises a nuclease activity that cuts the non-targetstrand at a first location, and/or cuts the target strand at a secondlocation. Depending on the nuclease activity, the target DNA can be cutto form a “double-stranded break” whereby both strands are cut. In otherembodiments, the target DNA can be cut at only a single site, i.e., theDNA is “nicked” on one strand. Exemplary napDNAbp with differentnuclease activities include “Cas9 nickase” (“nCas9”) and a deactivatedCas9 having no nuclease activities (“dead Cas9” or “dCas9”). Exemplarysequences for these and other napDNAbp are provided herein.

Nickase

The term “nickase” refers to a Cas9 with one of the two nuclease domainsinactivated. This enzyme is capable of cleaving only one strand of atarget DNA.

Nuclear Localization Sequence (NLS)

The term “nuclear localization sequence” or “NLS” refers to an aminoacid sequence that promotes import of a protein into the cell nucleus,for example, by nuclear transport. Nuclear localization sequences areknown in the art and would be apparent to the skilled artisan. Forexample, NLS sequences are described in Plank et al., international PCTapplication, PCT/EP2000/01 1690, filed Nov. 23, 2000, published asWO/2001/038547 on May 31, 2001, the contents of which are incorporatedherein by reference for its disclosure of exemplary nuclear localizationsequences. In some embodiments, a NLS comprises the amino acid sequencePKKKRKV (SEQ ID NO: 16) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO:17).

Nucleic Acid Molecule

The term “nucleic acid,” as used herein, refers to a polymer ofnucleotides. The polymer may include natural nucleosides (i.e.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine,C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8oxoguanosine, 0(6) methylguanine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine,1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and2-thiocytidine), chemically modified bases, biologically modified bases(e.g., methylated bases), intercalated bases, modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose,and hexose), or modified phosphate groups (e.g., phosphorothioates and5′ N phosphoramidite linkages).

PEgRNA

As used herein, the terms “prime editing guide RNA” or “PEgRNA” or“extended guide RNA” refers to a specialized form of a guide RNA thathas been modified to include one or more additional sequences forimplementing the prime editing methods and compositions describedherein. As described herein, the prime editing guide RNA comprise one ormore “extended regions” of nucleic acid sequence. The extended regionsmay comprise, but are not limited to, single-stranded RNA or DNA.Further, the extended regions may occur at the 3′ end of a traditionalguide RNA. In other arrangements, the extended regions may occur at the5′ end of a traditional guide RNA. In still other arrangements, theextended region may occur at an intramolecular region of the traditionalguide RNA, for example, in the gRNA core region which associates and/orbinds to the napDNAbp. The extended region comprises a “DNA synthesistemplate” which encodes (by the polymerase of the prime editor) asingle-stranded DNA which, in turn, has been designed to be (a)homologous with the endogenous target DNA to be edited, and (b) whichcomprises at least one desired nucleotide change (e.g., a transition, atransversion, a deletion, or an insertion) to be introduced orintegrated into the endogenous target DNA. The extended region may alsocomprise other functional sequence elements, such as, but not limitedto, a “primer binding site” and a “spacer or linker” sequence, or otherstructural elements, such as, but not limited to aptamers, stem loops,hairpins, toe loops (e.g., a 3′ toeloop), or an RNA-protein recruitmentdomain (e.g., MS2 hairpin). As used herein the “primer binding site”comprises a sequence that hybridizes to a single-strand DNA sequencehaving a 3′ end generated from the nicked DNA of the R-loop.

In certain embodiments, the PEgRNAs are represented by FIG. 3A, whichshows a PEgRNA having a 5′ extension arm, a spacer, and a gRNA core. The5′ extension further comprises in the 5′ to 3′ direction a reversetranscriptase template, a primer binding site, and a linker. As shown,the reverse transcriptase template may also be referred to more broadlyas the “DNA synthesis template” where the polymerase of a prime editordescribed herein is not an RT, but another type of polymerase.

In certain other embodiments, the PEgRNAs are represented by FIG. 3B,which shows a PEgRNA having a 5′ extension arm, a spacer, and a gRNAcore. The 5′ extension further comprises in the 5′ to 3′ direction areverse transcriptase template, a primer binding site, and a linker. Asshown, the reverse transcriptase template may also be referred to morebroadly as the “DNA synthesis template” where the polymerase of a primeeditor described herein is not an RT, but another type of polymerase.

In still other embodiments, the PEgRNAs are represented by FIG. 3D,which shows a PEgRNA having in the 5′ to 3′ direction a spacer (1), agRNA core (2), and an extension arm (3). The extension arm (3) is at the3′ end of the PEgRNA. The extension arm (3) further comprises in the 5′to 3′ direction a “primer binding site” (A), an “edit template” (B), anda “homology arm” (C). The extension arm (3) may also comprise anoptional modifier region at the 3′ and 5′ ends, which may be the samesequences or different sequences. In addition, the 3′ end of the PEgRNAmay comprise a transcriptional terminator sequence. These sequenceelements of the PEgRNAs are further described and defined herein.

In still other embodiments, the PEgRNAs are represented by FIG. 3E,which shows a PEgRNA having in the 5′ to 3′ direction an extension arm(3), a spacer (1), and a gRNA core (2). The extension arm (3) is at the5′ end of the PEgRNA. The extension arm (3) further comprises in the 3′to 5′ direction a “primer binding site” (A), an “edit template” (B), anda “homology arm” (C). The extension arm (3) may also comprise anoptional modifier region at the 3′ and 5′ ends, which may be the samesequences or different sequences. The PEgRNAs may also comprise atranscriptional terminator sequence at the 3′ end. These sequenceelements of the PEgRNAs are further described and defined herein.

PE1

As used herein, “PE1” refers to a PE complex comprising a fusion proteincomprising Cas9(H840A) and a wild type MMLV RT having the followingstructure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]+a desired PEgRNA,wherein the PE fusion has the amino acid sequence of SEQ ID NO: 123,which is shown as follows;

(SEQ ID NO: 123) MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSKRTADGS EFEPKKKRKV KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ IDNO: 124), BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 126) 33-AMINO  ACID   LINKER  (SEQ ID NO: 127)M-MLV reverse transcriptase (SEQ ID NO: 128).

PE2

As used herein, “PE2” refers to a PE complex comprising a fusion proteincomprising Cas9(H840A) and a variant MMLV RT having the followingstructure:[NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330)(L603W)(T306K)(W313F)]+adesired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQID NO: 134, which is shown as follows:

(SEQ ID NO: 134) MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSKRTADGS EFEPKKKRKV KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ IDNO: 124), BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 137) 33-AMINO  ACID   LINKER  (SEQ ID NO: 127)M-MLV reverse transcriptase (SEQ ID NO: 139).

PE3

As used herein, “PE3” refers to PE2 plus a second-strand nicking guideRNA that complexes with the PE2 and introduces a nick in the non-editedDNA strand in order to induce preferential replacement of the editedstrand.

PE3b

As used herein, “PE3b” refers to PE3 but wherein the second-strandnicking guide RNA is designed for temporal control such that the secondstrand nick is not introduced until after the installation of thedesired edit. This is achieved by designing a gRNA with a spacersequence that matches only the edited strand, but not the originalallele. Using this strategy, referred to hereafter as PE3b, mismatchesbetween the protospacer and the unedited allele should disfavor nickingby the sgRNA until after the editing event on the PAM strand takesplace.

PE-Short

As used herein, “PE-short” refers to a PE construct that is fused to aC-terminally truncated reverse transcriptase, and has the followingamino acid sequence:

(SEQ ID NO: 765) MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQ HNCLDNSRLINSGGSKRTADGSEFEPKKKRKV KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ IDNO: 124), BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 157) 33-AMINO  ACID   LINKER   1  (SEQ ID NO: 127)M-MLV TRUNCATED REVERSE TRANSCRIPTASE (SEQ ID NO: 766)

Peptide Tag

The term “peptide tag” refers to a peptide amino acid sequence that isgenetically fused to a protein sequence to impart one or more functionsonto the proteins that facilitate the manipulation of the protein forvarious purposes, such as, visualization, purification, solubilization,and separation, etc. Peptide tags can include various types of tagscategorized by purpose or function, which may include “affinity tags”(to facilitate protein purification), “solubilization tags” (to assistin proper folding of proteins), “chromatography tags” (to alterchromatographic properties of proteins), “epitope tags” (to bind to highaffinity antibodies), “fluorescence tags” (to facilitate visualizationof proteins in a cell or in vitro).

Polymerase

As used herein, the term “polymerase” refers to an enzyme thatsynthesizes a nucleotide strand and which may be used in connection withthe prime editor systems described herein. The polymerase can be a“template-dependent” polymerase (i.e., a polymerase which synthesizes anucleotide strand based on the order of nucleotide bases of a templatestrand). The polymerase can also be a “template-independent” polymerase(i.e., a polymerase which synthesizes a nucleotide strand without therequirement of a template strand). A polymerase may also be furthercategorized as a “DNA polymerase” or an “RNA polymerase.” In variousembodiments, the prime editor system comprises a DNA polymerase. Invarious embodiments, the DNA polymerase can be a “DNA-dependent DNApolymerase” (i.e., whereby the template molecule is a strand of DNA). Insuch cases, the DNA template molecule can be a PEgRNA, wherein theextension arm comprises a strand of DNA. In such cases, the PEgRNA maybe referred to as a chimeric or hybrid PEgRNA which comprises an RNAportion (i.e., the guide RNA components, including the spacer and thegRNA core) and a DNA portion (i.e., the extension arm). In various otherembodiments, the DNA polymerase can be an “RNA-dependent DNA polymerase”(i.e., whereby the template molecule is a strand of RNA). In such cases,the PEgRNA is RNA, i.e., including an RNA extension. The term“polymerase” may also refer to an enzyme that catalyzes thepolymerization of nucleotide (i.e., the polymerase activity). Generally,the enzyme will initiate synthesis at the 3′-end of a primer annealed toa polynucleotide template sequence (e.g., such as a primer sequenceannealed to the primer binding site of a PEgRNA), and will proceedtoward the 5′ end of the template strand. A “DNA polymerase” catalyzesthe polymerization of deoxynucleotides. As used herein in reference to aDNA polymerase, the term DNA polymerase includes a “functional fragmentthereof”. A “functional fragment thereof” refers to any portion of awild-type or mutant DNA polymerase that encompasses less than the entireamino acid sequence of the polymerase and which retains the ability,under at least one set of conditions, to catalyze the polymerization ofa polynucleotide. Such a functional fragment may exist as a separateentity, or it may be a constituent of a larger polypeptide, such as afusion protein.

Prime Editing

As used herein, the term “prime editing” refers to a novel approach forgene editing using napDNAbps, a polymerase (e.g., a reversetranscriptase), and specialized guide RNAs that include a DNA synthesistemplate for encoding desired new genetic information (or deletinggenetic information) that is then incorporated into a target DNAsequence. Certain embodiments of prime editing are described in theembodiments of FIGS. 1A-1H and FIG. 72(a)-72(c), among other figures.

Prime editing represents an entirely new platform for genome editingthat is a versatile and precise genome editing method that directlywrites new genetic information into a specified DNA site using a nucleicacid programmable DNA binding protein (“napDNAbp”) working inassociation with a polymerase (i.e., in the form of a fusion protein orotherwise provided in trans with the napDNAbp), wherein the primeediting system is programmed with a prime editing (PE) guide RNA(“PEgRNA”) that both specifies the target site and templates thesynthesis of the desired edit in the form of a replacement DNA strand byway of an extension (either DNA or RNA) engineered onto a guide RNA(e.g., at the 5′ or 3′ end, or at an internal portion of a guide RNA).The replacement strand containing the desired edit (e.g., a singlenucleobase substitution) shares the same (or is homologous to) sequenceas the endogenous strand (immediately downstream of the nick site) ofthe target site to be edited (with the exception that it includes thedesired edit). Through DNA repair and/or replication machinery, theendogenous strand downstream of the nick site is replaced by the newlysynthesized replacement strand containing the desired edit. In somecases, prime editing may be thought of as a “search-and-replace” genomeediting technology since the prime editors, as described herein, notonly search and locate the desired target site to be edited, but at thesame time, encode a replacement strand containing a desired edit whichis installed in place of the corresponding target site endogenous DNAstrand. The prime editors of the present disclosure relate, in part, tothe discovery that the mechanism of target-primed reverse transcription(TPRT) or “prime editing” can be leveraged or adapted for conductingprecision CRISPR/Cas-based genome editing with high efficiency andgenetic flexibility (e.g., as depicted in various embodiments of FIGS.1A-1F). TPRT is naturally used by mobile DNA elements, such as mammaliannon-LTR retrotransposons and bacterial Group II introns^(28,29). Theinventors have herein used Cas protein-reverse transcriptase fusions orrelated systems to target a specific DNA sequence with a guide RNA,generate a single strand nick at the target site, and use the nicked DNAas a primer for reverse transcription of an engineered reversetranscriptase template that is integrated with the guide RNA. However,while the concept begins with prime editors that use reversetranscriptase as the DNA polymerase component, the prime editorsdescribed herein are not limited to reverse transcriptases but mayinclude the use of virtually any DNA polymerase. Indeed, while theapplication throughout may refer to prime editors with “reversetranscriptases,” it is set forth here that reverse transcriptases areonly one type of DNA polymerase that may work with prime editing. Thus,where ever the specification mentions a “reverse transcriptase,” theperson having ordinary skill in the art should appreciate that anysuitable DNA polymerase may be used in place of the reversetranscriptase. Thus, in one aspect, the prime editors may comprise Cas9(or an equivalent napDNAbp) which is programmed to target a DNA sequenceby associating it with a specialized guide RNA (i.e., PEgRNA) containinga spacer sequence that anneals to a complementary protospacer in thetarget DNA. The specialized guide RNA also contains new geneticinformation in the form of an extension that encodes a replacementstrand of DNA containing a desired genetic alteration which is used toreplace a corresponding endogenous DNA strand at the target site. Totransfer information from the PEgRNA to the target DNA, the mechanism ofprime editing involves nicking the target site in one strand of the DNAto expose a 3′-hydroxyl group. The exposed 3′-hydroxyl group can then beused to prime the DNA polymerization of the edit-encoding extension onPEgRNA directly into the target site. In various embodiments, theextension-which provides the template for polymerization of thereplacement strand containing the edit—can be formed from RNA or DNA. Inthe case of an RNA extension, the polymerase of the prime editor can bean RNA-dependent DNA polymerase (such as, a reverse transcriptase). Inthe case of a DNA extension, the polymerase of the prime editor may be aDNA-dependent DNA polymerase. The newly synthesized strand (i.e., thereplacement DNA strand containing the desired edit) that is formed bythe herein disclosed prime editors would be homologous to the genomictarget sequence (i.e., have the same sequence as) except for theinclusion of a desired nucleotide change (e.g., a single nucleotidechange, a deletion, or an insertion, or a combination thereof). Thenewly synthesized (or replacement) strand of DNA may also be referred toas a single strand DNA flap, which would compete for hybridization withthe complementary homologous endogenous DNA strand, thereby displacingthe corresponding endogenous strand. In certain embodiments, the systemcan be combined with the use of an error-prone reverse transcriptaseenzyme (e.g., provided as a fusion protein with the Cas9 domain, orprovided in trans to the Cas9 domain). The error-prone reversetranscriptase enzyme can introduce alterations during synthesis of thesingle strand DNA flap. Thus, in certain embodiments, error-pronereverse transcriptase can be utilized to introduce nucleotide changes tothe target DNA. Depending on the error-prone reverse transcriptase thatis used with the system, the changes can be random or non-random.Resolution of the hybridized intermediate (comprising the single strandDNA flap synthesized by the reverse transcriptase hybridized to theendogenous DNA strand) can include removal of the resulting displacedflap of endogenous DNA (e.g., with a 5′ end DNA flap endonuclease,FEN1), ligation of the synthesized single strand DNA flap to the targetDNA, and assimilation of the desired nucleotide change as a result ofcellular DNA repair and/or replication processes. Because templated DNAsynthesis offers single nucleotide precision for the modification of anynucleotide, including insertions and deletions, the scope of thisapproach is very broad and could foreseeably be used for myriadapplications in basic science and therapeutics.

In various embodiments, prime editing operates by contacting a targetDNA molecule (for which a change in the nucleotide sequence is desiredto be introduced) with a nucleic acid programmable DNA binding protein(napDNAbp) complexed with a prime editing guide RNA (PEgRNA). Inreference to FIG. 1G, the prime editing guide RNA (PEgRNA) comprises anextension at the 3′ or 5′ end of the guide RNA, or at an intramolecularlocation in the guide RNA and encodes the desired nucleotide change(e.g., single nucleotide change, insertion, or deletion). In step (a),the napDNAbp/extended gRNA complex contacts the DNA molecule and theextended gRNA guides the napDNAbp to bind to a target locus. In step(b), a nick in one of the strands of DNA of the target locus isintroduced (e.g., by a nuclease or chemical agent), thereby creating anavailable 3′ end in one of the strands of the target locus. In certainembodiments, the nick is created in the strand of DNA that correspondsto the R-loop strand, i.e., the strand that is not hybridized to theguide RNA sequence, i.e., the “non-target strand.” The nick, however,could be introduced in either of the strands. That is, the nick could beintroduced into the R-loop “target strand” (i.e., the strand hybridizedto the protospacer of the extended gRNA) or the “non-target strand”(i.e., the strand forming the single-stranded portion of the R-loop andwhich is complementary to the target strand). In step (c), the 3′ end ofthe DNA strand (formed by the nick) interacts with the extended portionof the guide RNA in order to prime reverse transcription (i.e.,“target-primed RT”). In certain embodiments, the 3′ end DNA strandhybridizes to a specific RT priming sequence on the extended portion ofthe guide RNA, i.e., the “reverse transcriptase priming sequence” or“primer binding site” on the PEgRNA. In step (d), a reversetranscriptase (or other suitable DNA polymerase) is introduced whichsynthesizes a single strand of DNA from the 3′ end of the primed sitetowards the 5′ end of the prime editing guide RNA. The DNA polymerase(e.g., reverse transcriptase) can be fused to the napDNAbp oralternatively can be provided in trans to the napDNAbp. This forms asingle-strand DNA flap comprising the desired nucleotide change (e.g.,the single base change, insertion, or deletion, or a combinationthereof) and which is otherwise homologous to the endogenous DNA at oradjacent to the nick site. In step (e), the napDNAbp and guide RNA arereleased. Steps (f) and (g) relate to the resolution of the singlestrand DNA flap such that the desired nucleotide change becomesincorporated into the target locus. This process can be driven towardsthe desired product formation by removing the corresponding 5′endogenous DNA flap that forms once the 3′ single strand DNA flapinvades and hybridizes to the endogenous DNA sequence. Without beingbound by theory, the cells endogenous DNA repair and replicationprocesses resolves the mismatched DNA to incorporate the nucleotidechange(s) to form the desired altered product. The process can also bedriven towards product formation with “second strand nicking,” asexemplified in FIG. 1F. This process may introduce at least one or moreof the following genetic changes: transversions, transitions, deletions,and insertions.

The term “prime editor (PE) system” or “prime editor (PE)” or “PEsystem” or “PE editing system” refers the compositions involved in themethod of genome editing using target-primed reverse transcription(TPRT) describe herein, including, but not limited to the napDNAbps,reverse transcriptases, fusion proteins (e.g., comprising napDNAbps andreverse transcriptases), prime editing guide RNAs, and complexescomprising fusion proteins and prime editing guide RNAs, as well asaccessory elements, such as second strand nicking components (e.g.,second strand sgRNAs) and 5′ endogenous DNA flap removal endonucleases(e.g., FEN1) for helping to drive the prime editing process towards theedited product formation.

Although in the embodiments described thus far the PEgRNA constitutes asingle molecule comprising a guide RNA (which itself comprises a spacersequence and a gRNA core or scaffold) and a 5′ or 3′ extension armcomprising the primer binding site and a DNA synthesis template (e.g.,see FIG. 3D, the PEgRNA may also take the form of two individualmolecules comprised of a guide RNA and a trans prime editor RNA template(tPERT), which essentially houses the extension arm (including, inparticular, the primer binding site and the DNA synthesis domain) and anRNA-protein recruitment domain (e.g., MS2 aptamer or hairpin) in thesame molecule which becomes co-localized or recruited to a modifiedprime editor complex that comprises a tPERT recruiting protein (e.g.,MS2cp protein, which binds to the MS2 aptamer). See FIG. 3G and FIG. 3Has an example of a tPERT that may be used with prime editing.

Prime Editor

The term “prime editor” refers to the herein described fusion constructscomprising a napDNAbp (e.g., Cas9 nickase) and a reverse transcriptaseand is capable of carrying out prime editing on a target nucleotidesequence in the presence of a PEgRNA (or “extended guide RNA”). The term“prime editor” may refer to the fusion protein or to the fusion proteincomplexed with a PEgRNA, and/or further complexed with a second-strandnicking sgRNA. In some embodiments, the prime editor may also refer tothe complex comprising a fusion protein (reverse transcriptase fused toa napDNAbp), a PEgRNA, and a regular guide RNA capable of directing thesecond-site nicking step of the non-edited strand as described herein.In other embodiments, the reverse transcriptase component of the “primereditor” may be provided in trans.

Primer Binding Site

The term “primer binding site” or “the PBS” refers to the nucleotidesequence located on a PEgRNA as component of the extension arm(typically at the 3′ end of the extension arm) and serves to bind to theprimer sequence that is formed after Cas9 nicking of the target sequenceby the prime editor. As detailed elsewhere, when the Cas9 nickasecomponent of a prime editor nicks one strand of the target DNA sequence,a 3′-ended ssDNA flap is formed, which serves a primer sequence thatanneals to the primer binding site on the PEgRNA to prime reversetranscription. FIGS. 27 and 28 show embodiments of the primer bindingsite located on a 3′ and 5′ extension arm, respectively.

Promoter

The term “promoter” is art-recognized and refers to a nucleic acidmolecule with a sequence recognized by the cellular transcriptionmachinery and able to initiate transcription of a downstream gene. Apromoter can be constitutively active, meaning that the promoter isalways active in a given cellular context, or conditionally active,meaning that the promoter is only active in the presence of a specificcondition. For example, a conditional promoter may only be active in thepresence of a specific protein that connects a protein associated with aregulatory element in the promoter to the basic transcriptionalmachinery, or only in the absence of an inhibitory molecule. A subclassof conditionally active promoters are inducible promoters that requirethe presence of a small molecule “inducer” for activity. Examples ofinducible promoters include, but are not limited to, arabinose-induciblepromoters, Tet-on promoters, and tamoxifen-inducible promoters. Avariety of constitutive, conditional, and inducible promoters are wellknown to the skilled artisan, and the skilled artisan will be able toascertain a variety of such promoters useful in carrying out the instantinvention, which is not limited in this respect.

Protospacer

As used herein, the term “protospacer” refers to the sequence (˜20 bp)in DNA adjacent to the PAM (protospacer adjacent motif) sequence. Theprotospacer shares the same sequence as the spacer sequence of the guideRNA. The guide RNA anneals to the complement of the protospacer sequenceon the target DNA (specifically, one strand thereof, i.e., the “targetstrand” versus the “non-target strand” of the target DNA sequence). Inorder for Cas9 to function it also requires a specific protospaceradjacent motif (PAM) that varies depending on the bacterial species ofthe Cas9 gene. The most commonly used Cas9 nuclease, derived from S.pyogenes, recognizes a PAM sequence of NGG that is found directlydownstream of the target sequence in the genomic DNA, on the non-targetstrand. The skilled person will appreciate that the literature in thestate of the art sometimes refers to the “protospacer” as the ˜20-nttarget-specific guide sequence on the guide RNA itself, rather thanreferring to it as a “spacer.” Thus, in some cases, the term“protospacer” as used herein may be used interchangeably with the term“spacer.” The context of the description surrounding the appearance ofeither “protospacer” or “spacer” will help inform the reader as towhether the term is in reference to the gRNA or the DNA target.

Protospacer Adjacent Motif (PAM)

As used herein, the term “protospacer adjacent sequence” or “PAM” refersto an approximately 2-6 base pair DNA sequence that is an importanttargeting component of a Cas9 nuclease. Typically, the PAM sequence ison either strand, and is downstream in the 5′ to 3′ direction of Cas9cut site. The canonical PAM sequence (i.e., the PAM sequence that isassociated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9)is 5′-NGG-3′ wherein “N” is any nucleobase followed by two guanine (“G”)nucleobases. Different PAM sequences can be associated with differentCas9 nucleases or equivalent proteins from different organisms. Inaddition, any given Cas9 nuclease, e.g., SpCas9, may be modified toalter the PAM specificity of the nuclease such that the nucleaserecognizes alternative PAM sequence.

For example, with reference to the canonical SpCas9 amino acid sequenceis SEQ ID NO: 18, the PAM sequence can be modified by introducing one ormore mutations, including (a) D1135V, R1335Q, and T1337R “the VQRvariant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E,R1335Q, and T1337R “the EQR variant”, which alters the PAM specificityto NGAG, and (c) D1135V, G1218R, R1335E, and T1337R “the VRER variant”,which alters the PAM specificity to NGCG. In addition, the D1135Evariant of canonical SpCas9 still recognizes NGG, but it is moreselective compared to the wild type SpCas9 protein.

It will also be appreciated that Cas9 enzymes from different bacterialspecies (i.e., Cas9 orthologs) can have varying PAM specificities. Forexample, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT orNGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizesNNNNGATT. In another example, Cas9 from Streptococcus thermophilis(StCas9) recognizes NNAGAAW. In still another example, Cas9 fromTreponema denticola (TdCas) recognizes NAAAAC. These are example are notmeant to be limiting. It will be further appreciated that non-SpCas9sbind a variety of PAM sequences, which makes them useful when nosuitable SpCas9 PAM sequence is present at the desired target cut site.Furthermore, non-SpCas9s may have other characteristics that make themmore useful than SpCas9. For example, Cas9 from Staphylococcus aureus(SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packagedinto adeno-associated virus (AAV). Further reference may be made to Shahet al., “Protospacer recognition motifs: mixed identities and functionaldiversity,” RNA Biology, 10(5): 891-899 (which is incorporated herein byreference).

Recombinase

The term “recombinase,” as used herein, refers to a site-specific enzymethat mediates the recombination of DNA between recombinase recognitionsequences, which results in the excision, integration, inversion, orexchange (e.g., translocation) of DNA fragments between the recombinaserecognition sequences. Recombinases can be classified into two distinctfamilies: serine recombinases (e.g., resolvases and invertases) andtyrosine recombinases (e.g., integrases). Examples of serinerecombinases include, without limitation, Hin, Gin, Tn3, β-six, CinH,ParA, γδ, Bxb1, ϕC31, TP901, TG1, φBT1, R4, φRV1, φFC1, MR11, A118,U153, and gp29. Examples of tyrosine recombinases include, withoutlimitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The serine andtyrosine recombinase names stem from the conserved nucleophilic aminoacid residue that the recombinase uses to attack the DNA and whichbecomes covalently linked to the DNA during strand exchange.Recombinases have numerous applications, including the creation of geneknockouts/knock-ins and gene therapy applications. See, e.g., Brown etal., “Serine recombinases as tools for genome engineering.” Methods.2011; 53(4):372-9; Hirano et al., “Site-specific recombinases as toolsfor heterologous gene integration.” Appl. Microbiol. Biotechnol. 2011;92(2):227-39; Chavez and Calos, “Therapeutic applications of the ΦC31integrase system.” Curr. Gene Ther. 2011; 11(5):375-81; Turan and Bode,“Site-specific recombinases: from tag-and-target-totag-and-exchange-based genomic modifications.” FASEB J. 2011;25(12):4088-107; Venken and Bellen, “Genome-wide manipulations ofDrosophila melanogaster with transposons, Flp recombinase, and ΦC31integrase.” Methods Mol. Biol. 2012; 859:203-28; Murphy, “Phagerecombinases and their applications.” Adv. Virus Res. 2012; 83:367-414;Zhang et al., “Conditional gene manipulation: Cre-ating a new biologicalera.” J. Zhejiang Univ. Sci. B. 2012; 13(7):511-24; Karpenshif andBernstein, “From yeast to mammals: recent advances in genetic control ofhomologous recombination.” DNA Repair (Amst). 2012; 1; 11(10):781-8; theentire contents of each are hereby incorporated by reference in theirentirety. The recombinases provided herein are not meant to be exclusiveexamples of recombinases that can be used in embodiments of theinvention. The methods and compositions of the invention can be expandedby mining databases for new orthogonal recombinases or designingsynthetic recombinases with defined DNA specificities (See, e.g., Grothet al., “Phage integrases: biology and applications.” J. Mol. Biol.2004; 335, 667-678; Gordley et al., “Synthesis of programmableintegrases.” Proc. Natl. Acad. Sci. USA. 2009; 106, 5053-5058; theentire contents of each are hereby incorporated by reference in theirentirety). Other examples of recombinases that are useful in the methodsand compositions described herein are known to those of skill in theart, and any new recombinase that is discovered or generated is expectedto be able to be used in the different embodiments of the invention. Insome embodiments, the catalytic domains of a recombinase are fused to anuclease-inactivated RNA-programmable nuclease (e.g., dCas9, or afragment thereof), such that the recombinase domain does not comprise anucleic acid binding domain or is unable to bind to a target nucleicacid (e.g., the recombinase domain is engineered such that it does nothave specific DNA binding activity). Recombinases lacking DNA bindingactivity and methods for engineering such are known, and include thosedescribed by Klippel et al., “Isolation and characterisation of unusualgin mutants.” EMBO J. 1988; 7: 3983-3989: Burke et al., “Activatingmutations of Tn3 resolvase marking interfaces important in recombinationcatalysis and its regulation. Mol Microbiol. 2004; 51: 937-948;Olorunniji et al., “Synapsis and catalysis by activated Tn3 resolvasemutants.” Nucleic Acids Res. 2008; 36: 7181-7191; Rowland et al.,“Regulatory mutations in Sin recombinase support a structure-based modelof the synaptosome.” Mol Microbiol. 2009; 74: 282-298; Akopian et al.,“Chimeric recombinases with designed DNA sequence recognition.” ProcNatl Acad Sci USA. 2003; 100: 8688-8691; Gordley et al., “Evolution ofprogrammable zinc finger-recombinases with activity in human cells. JMol Biol. 2007; 367: 802-813; Gordley et al., “Synthesis of programmableintegrases.” Proc Natl Acad Sci USA. 2009; 106: 5053-5058; Arnold etal., “Mutants of Tn3 resolvase which do not require accessory bindingsites for recombination activity.” EMBO J. 1999; 18: 1407-1414; Gaj etal., “Structure-guided reprogramming of serine recombinase DNA sequencespecificity.” Proc Natl Acad Sci USA. 2011; 108(2):498-503; andProudfoot et al., “Zinc finger recombinases with adaptable DNA sequencespecificity.” PLoS One. 2011; 6(4):e19537; the entire contents of eachare hereby incorporated by reference. For example, serine recombinasesof the resolvase-invertase group, e.g., Tn3 and γδ resolvases and theHin and Gin invertases, have modular structures with autonomouscatalytic and DNA-binding domains (See, e.g., Grindley et al.,“Mechanism of site-specific recombination.” Ann Rev Biochem. 2006; 75:567-605, the entire contents of which are incorporated by reference).The catalytic domains of these recombinases are thus amenable to beingrecombined with nuclease-inactivated RNA-programmable nucleases (e.g.,dCas9, or a fragment thereof) as described herein, e.g., following theisolation of ‘activated’ recombinase mutants which do not require anyaccessory factors (e.g., DNA binding activities) (See, e.g., Klippel etal., “Isolation and characterisation of unusual gin mutants.” EMBO J.1988; 7: 3983-3989: Burke et al., “Activating mutations of Tn3 resolvasemarking interfaces important in recombination catalysis and itsregulation. Mol Microbiol. 2004; 51: 937-948; Olorunniji et al.,“Synapsis and catalysis by activated Tn3 resolvase mutants.” NucleicAcids Res. 2008; 36: 7181-7191; Rowland et al., “Regulatory mutations inSin recombinase support a structure-based model of the synaptosome.” MolMicrobiol. 2009; 74: 282-298; Akopian et al., “Chimeric recombinaseswith designed DNA sequence recognition.” Proc Natl Acad Sci USA. 2003;100: 8688-8691). Additionally, many other natural serine recombinaseshaving an N-terminal catalytic domain and a C-terminal DNA bindingdomain are known (e.g., phiC31 integrase, TnpX transposase, IS607transposase), and their catalytic domains can be co-opted to engineerprogrammable site-specific recombinases as described herein (See, e.g.,Smith et al., “Diversity in the serine recombinases.” Mol Microbiol.2002; 44: 299-307, the entire contents of which are incorporated byreference). Similarly, the core catalytic domains of tyrosinerecombinases (e.g., Cre, λ integrase) are known, and can be similarlyco-opted to engineer programmable site-specific recombinases asdescribed herein (See, e.g., Guo et al., “Structure of Cre recombinasecomplexed with DNA in a site-specific recombination synapse.” Nature.1997; 389:40-46; Hartung et al., “Cre mutants with altered DNA bindingproperties.” J Biol Chem 1998; 273:22884-22891; Shaikh et al., “Chimerasof the Flp and Cre recombinases: Tests of the mode of cleavage by Flpand Cre. J Mol Biol. 2000; 302:27-48; Rongrong et al., “Effect ofdeletion mutation on the recombination activity of Cre recombinase.”Acta Biochim Pol. 2005; 52:541-544; Kilbride et al., “Determinants ofproduct topology in a hybrid Cre-Tn3 resolvase site-specificrecombination system.” J Mol Biol. 2006; 355:185-195; Warren et al., “Achimeric cre recombinase with regulated directionality.” Proc Natl AcadSci USA. 2008 105:18278-18283; Van Duyne, “Teaching Cre to followdirections.” Proc Natl Acad Sci USA. 2009 Jan. 6; 106(1):4-5; Numrych etal., “A comparison of the effects of single-base and triple-base changesin the integrase arm-type binding sites on the site-specificrecombination of bacteriophage λ.” Nucleic Acids Res. 1990;18:3953-3959; Tirumalai et al., “The recognition of core-type DNA sitesby λ integrase.” J Mol Biol. 1998; 279:513-527; Aihara et al., “Aconformational switch controls the DNA cleavage activity of λintegrase.” Mol Cell. 2003; 12:187-198; Biswas et al., “A structuralbasis for allosteric control of DNA recombination by λ integrase.”Nature. 2005; 435:1059-1066; and Warren et al., “Mutations in theamino-terminal domain of λ-integrase have differential effects onintegrative and excisive recombination.” Mol Microbiol. 2005;55:1104-1112; the entire contents of each are incorporated byreference).

Recombinase Recognition Sequence

The term “recombinase recognition sequence”, or equivalently as “RRS” or“recombinase target sequence”, as used herein, refers to a nucleotidesequence target recognized by a recombinase and which undergoes strandexchange with another DNA molecule having a the RRS that results inexcision, integration, inversion, or exchange of DNA fragments betweenthe recombinase recognition sequences.

Recombine or Recombination

The term “recombine,” or “recombination,” in the context of a nucleicacid modification (e.g., a genomic modification), is used to refer tothe process by which two or more nucleic acid molecules, or two or moreregions of a single nucleic acid molecule, are modified by the action ofa recombinase protein (e.g., an inventive recombinase fusion proteinprovided herein). Recombination can result in, inter alia, theinsertion, inversion, excision, or translocation of nucleic acids, e.g.,in or between one or more nucleic acid molecules. recombinaserecognition sequences

Reverse Transcriptase

The term “reverse transcriptase” describes a class of polymerasescharacterized as RNA-dependent DNA polymerases. All known reversetranscriptases require a primer to synthesize a DNA transcript from anRNA template. Historically, reverse transcriptase has been usedprimarily to transcribe mRNA into cDNA which can then be cloned into avector for further manipulation. Avian myoblastosis virus (AMV) reversetranscriptase was the first widely used RNA-dependent DNA polymerase(Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme has 5′-3′RNA-directed DNA polymerase activity, 5′-3′ DNA-directed DNA polymeraseactivity, and RNase H activity. RNase H is a processive 5′ and 3′ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, APractical Guide to Molecular Cloning, New York: Wiley & Sons (1984)).Errors in transcription cannot be corrected by reverse transcriptasebecause known viral reverse transcriptases lack the 3′-5′ exonucleaseactivity necessary for proofreading (Saunders and Saunders, MicrobialGenetics Applied to Biotechnology, London: Croom Helm (1987)). Adetailed study of the activity of AMV reverse transcriptase and itsassociated RNase H activity has been presented by Berger et al.,Biochemistry 22:2365-2372 (1983). Another reverse transcriptase which isused extensively in molecular biology is reverse transcriptaseoriginating from Moloney murine leukemia virus (M-MLV). See, e.g.,Gerard, G. R., DNA 5:271-279 (1986) and Kotewicz, M. L., et al., Gene35:249-258 (1985). M-MLV reverse transcriptase substantially lacking inRNase H activity has also been described. See, e.g., U.S. Pat. No.5,244,797. The invention contemplates the use of any such reversetranscriptases, or variants or mutants thereof.

In addition, the invention contemplates the use of reversetranscriptases which are error-prone, i.e., which may be referred to aserror-prone reverse transcriptases or reverse transcriptases which donot support high fidelity incorporation of nucleotides duringpolymerization. During synthesis of the single-strand DNA flap based onthe RT template integrated with the guide RNA, the error-prone reversetranscriptase can introduce one or more nucleotides which are mismatchedwith the RT template sequence, thereby introducing changes to thenucleotide sequence through erroneous polymerization of thesingle-strand DNA flap. These errors introduced during synthesis of thesingle strand DNA flap then become integrated into the double strandmolecule through hybridization to the corresponding endogenous targetstrand, removal of the endogenous displaced strand, ligation, and thenthrough one more round of endogenous DNA repair and/or sequencingprocesses.

Reverse Transcription

As used herein, the term “reverse transcription” indicates thecapability of enzyme to synthesize DNA strand (that is, complementaryDNA or cDNA) using RNA as a template. In some embodiments, the reversetranscription can be “error-prone reverse transcription,” which refersto the properties of certain reverse transcriptase enzymes which areerror-prone in their DNA polymerization activity.

PACE

The term “phage-assisted continuous evolution (PACE),” as used herein,refers to continuous evolution that employs phage as viral vectors. Thegeneral concept of PACE technology has been described, for example, inInternational PCT application, PCT/US2009/056194, filed Sep. 8, 2009,published as WO 2010/028347 on Mar. 11, 2010; International PCTapplication, PCT/US2011/066747, filed Dec. 22, 2011, published as WO2012/088381 on Jun. 28, 2012; U.S. application, U.S. Pat. No. 9,023,594,issued May 5, 2015, International PCT application, PCT/US2015/012022,filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015, andInternational PCT application, PCT/US2016/027795, filed Apr. 15, 2016,published as WO 2016/168631 on Oct. 20, 2016, the entire contents ofeach of which are incorporated herein by reference.

Page

The term “phage,” as used herein interchangeably with the term“bacteriophage,” refers to a virus that infects bacterial cells.Typically, phages consist of an outer protein capsid enclosing geneticmaterial. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA, ineither linear or circular form. Phages and phage vectors are well knownto those of skill in the art and non-limiting examples of phages thatare useful for carrying out the PACE methods provided herein are λ(Lysogen), T2, T4, T7, T12, R17, M13, MS2, G4, P1, P2, P4, Phi X174, N4,Φ6, and Φ29. In certain embodiments, the phage utilized in the presentinvention is M13. Additional suitable phages and host cells will beapparent to those of skill in the art and the invention is not limitedin this aspect. For an exemplary description of additional suitablephages and host cells, see Elizabeth Kutter and Alexander Sulakvelidze:Bacteriophages: Biology and Applications. CRC Press; 1st edition(December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M.Kropinski: Bacteriophages: Methods and Protocols, Volume 1: Isolation,Characterization, and Interactions (Methods in Molecular Biology) HumanaPress; 1st edition (December, 2008), ISBN: 1588296822; Martha R. J.Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols,Volume 2: Molecular and Applied Aspects (Methods in Molecular Biology)Humana Press; 1st edition (December 2008), ISBN: 1603275649; all ofwhich are incorporated herein in their entirety by reference fordisclosure of suitable phages and host cells as well as methods andprotocols for isolation, culture, and manipulation of such phages).

Protein, Peptide, and Polypeptide

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably herein, and refer to a polymer of amino acid residueslinked together by peptide (amide) bonds. The terms refer to a protein,peptide, or polypeptide of any size, structure, or function. Typically,a protein, peptide, or polypeptide will be at least three amino acidslong. A protein, peptide, or polypeptide may refer to an individualprotein or a collection of proteins. One or more of the amino acids in aprotein, peptide, or polypeptide may be modified, for example, by theaddition of a chemical entity such as a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. A protein, peptide, or polypeptide may also be asingle molecule or may be a multi-molecular complex. A protein, peptide,or polypeptide may be just a fragment of a naturally occurring proteinor peptide. A protein, peptide, or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof. Any ofthe proteins provided herein may be produced by any method known in theart. For example, the proteins provided herein may be produced viarecombinant protein expression and purification, which is especiallysuited for fusion proteins comprising a peptide linker. Methods forrecombinant protein expression and purification are well known, andinclude those described by Green and Sambrook, Molecular Cloning: ALaboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2012)), the entire contents of which areincorporated herein by reference.

Protein Splicing

The term “protein splicing,” as used herein, refers to a process inwhich a sequence, an intein (or split inteins, as the case may be), isexcised from within an amino acid sequence, and the remaining fragmentsof the amino acid sequence, the exteins, are ligated via an amide bondto form a continuous amino acid sequence. The term “trans” proteinsplicing refers to the specific case where the inteins are split inteinsand they are located on different proteins.

Second-Strand Nicking

The resolution of heteroduplex DNA (i.e., containing one edited and onenon-edited strand) formed as a result of prime editing determineslong-term editing outcomes. In words, a goal of prime editing is toresolve the heteroduplex DNA (the edited strand paired with theendogenous non-edited strand) formed as an intermediate of PE bypermanently integrating the edited strand into the complement,endogenous strand. The approach of “second-strand nicking” can be usedherein to help drive the resolution of heteroduplex DNA in favor ofpermanent integration of the edited strand into the DNA molecule. Asused herein, the concept of “second-strand nicking” refers to theintroduction of a second nick at a location downstream of the first nick(i.e., the initial nick site that provides the free 3′ end for use inpriming of the reverse transcriptase on the extended portion of theguide RNA), preferably on the unedited strand. In certain embodiments,the first nick and the second nick are on opposite strands. In otherembodiments, the first nick and the second nick are on opposite strands.In yet another embodiment, the first nick is on the non-target strand(i.e., the strand that forms the single strand portion of the R-loop),and the second nick is on the target strand. In still other embodiments,the first nick is on the edited strand, and the second nick is on theunedited strand. The second nick can be positioned at least 5nucleotides downstream of the first nick, or at least 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 or morenucleotides downstream of the first nick. The second nick, in certainembodiments, can be introduced between about 5-150 nucleotides on theunedited strand away from the site of the PEgRNA-induced nick, orbetween about 5-140, or between about 5-130, or between about 5-120, orbetween about 5-110, or between about 5-100, or between about 5-90, orbetween about 5-80, or between about 5-70, or between about 5-60, orbetween about 5-50, or between about 5-40, or between about 5-30, orbetween about 5-20, or between about 5-10. In one embodiment, the secondnick is introduced between 14-116 nucleotides away from thePEgRNA-induced nick. Without being bound by theory, the second nickinduces the cell's endogenous DNA repair and replication processestowards replacement or editing of the unedited strand, therebypermanently installing the edited sequence on both strands and resolvingthe heteroduplex that is formed as a result of PE. In some embodiments,the edited strand is the non-target strand and the unedited strand isthe target strand. In other embodiments, the edited strand is the targetstrand, and the unedited strand is the non-target strand.

Sense Strand

In genetics, a “sense” strand is the segment within double-stranded DNAthat runs from 5′ to 3′, and which is complementary to the antisensestrand of DNA, or template strand, which runs from 3′ to 5′. In the caseof a DNA segment that encodes a protein, the sense strand is the strandof DNA that has the same sequence as the mRNA, which takes the antisensestrand as its template during transcription, and eventually undergoes(typically, not always) translation into a protein. The antisense strandis thus responsible for the RNA that is later translated to protein,while the sense strand possesses a nearly identical makeup to that ofthe mRNA. Note that for each segment of dsDNA, there will possibly betwo sets of sense and antisense, depending on which direction one reads(since sense and antisense is relative to perspective). It is ultimatelythe gene product, or mRNA, that dictates which strand of one segment ofdsDNA is referred to as sense or antisense.

In the context of a PEgRNA, the first step is the synthesis of asingle-strand complementary DNA (i.e., the 3′ ssDNA flap, which becomesincorporated) oriented in the 5′ to 3′ direction which is templated offof the PEgRNA extension arm. Whether the 3′ ssDNA flap should beregarded as a sense or antisense strand depends on the direction oftranscription since it well accepted that both strands of DNA may serveas a template for transcription (but not at the same time). Thus, insome embodiments, the 3′ ssDNA flap (which overall runs in the 5′ to 3′direction) will serve as the sense strand because it is the codingstrand. In other embodiments, the 3′ ssDNA flap (which overall runs inthe 5′ to 3′ direction) will serve as the antisense strand and thus, thetemplate for transcription.

Spacer Sequence

As used herein, the term “spacer sequence” in connection with a guideRNA or a PEgRNA refers to the portion of the guide RNA or PEgRNA ofabout 20 nucleotides which contains a nucleotide sequence that iscomplementary to the protospacer sequence in the target DNA sequence.The spacer sequence anneals to the protospacer sequence to form assRNA/ssDNA hybrid structure at the target site and a corresponding Rloop ssDNA structure of the endogenous DNA strand that is complementaryto the protospacer sequence.

Subject

The term “subject,” as used herein, refers to an individual organism,for example, an individual mammal. In some embodiments, the subject is ahuman. In some embodiments, the subject is a non-human mammal. In someembodiments, the subject is a non-human primate. In some embodiments,the subject is a rodent. In some embodiments, the subject is a sheep, agoat, a cattle, a cat, or a dog. In some embodiments, the subject is avertebrate, an amphibian, a reptile, a fish, an insect, a fly, or anematode. In some embodiments, the subject is a research animal. In someembodiments, the subject is genetically engineered, e.g., a geneticallyengineered non-human subject. The subject may be of either sex and atany stage of development.

Split Intein

Although inteins are most frequently found as a contiguous domain, someexist in a naturally split form. In this case, the two fragments areexpressed as separate polypeptides and must associate before splicingtakes place, so-called protein trans-splicing.

An exemplary split intein is the Ssp DnaE intein, which comprises twosubunits, namely, DnaE-N and DnaE-C. The two different subunits areencoded by separate genes, namely dnaE-n and dnaE-c, which encode theDnaE-N and DnaE-C subunits, respectively. DnaE is a naturally occurringsplit intein in Synechocystis sp. PCC6803 and is capable of directingtrans-splicing of two separate proteins, each comprising a fusion witheither DnaE-N or DnaE-C.

Additional naturally occurring or engineered split-intein sequences areknown in the or can be made from whole-intein sequences described hereinor those available in the art. Examples of split-intein sequences can befound in Stevens et al., “A promiscuous split intein with expandedprotein engineering applications,” PNAS, 2017, Vol. 114: 8538-8543; Iwaiet al., “Highly efficient protein trans-splicing by a naturally splitDnaE intein from Nostoc punctiforme, FEBS Lett, 580: 1853-1858, each ofwhich are incorporated herein by reference. Additional split inteinsequences can be found, for example, in WO 2013/045632, WO 2014/055782,WO 2016/069774, and EP2877490, the contents each of which areincorporated herein by reference.

In addition, protein splicing in trans has been described in vivo and invitro (Shingledecker, et al., Gene 207:187 (1998), Southworth, et al.,EMBO J. 17:918 (1998); Mills, et al., Proc. Natl. Acad. Sci. USA,95:3543-3548 (1998); Lew, et al., J. Biol. Chem., 273:15887-15890(1998); Wu, et al., Biochim. Biophys. Acta 35732:1 (1998b), Yamazaki, etal., J. Am. Chem. Soc. 120:5591 (1998), Evans, et al., J. Biol. Chem.275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999);Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc.Natl. Acad. Sci. USA 96:13638-13643 (1999)) and provides the opportunityto express a protein as to two inactive fragments that subsequentlyundergo ligation to form a functional product, e.g., as shown in FIGS.66 and 67 with regard to the formation of a complete PE fusion proteinfrom two separately-expressed halves.

Target Site

The term “target site” refers to a sequence within a nucleic acidmolecule that is edited by a prime editor (PE) disclosed herein. Thetarget site further refers to the sequence within a nucleic acidmolecule to which a complex of the prime editor (PE) and gRNA binds.

tPERT

See definition for “trans prime editor RNA template (tPERT).”

Temporal Second-Strand Nicking

As used herein, the term “temporal second-strand nicking” refers to avariant of second strand nicking whereby the installation of the secondnick in the unedited strand occurs only after the desired edit isinstalled in the edited strand. This avoids concurrent nicks on bothstrands that could lead to double-stranded DNA breaks. The second-strandnicking guide RNA is designed for temporal control such that the secondstrand nick is not introduced until after the installation of thedesired edit. This is achieved by designing a gRNA with a spacersequence that matches only the edited strand, but not the originalallele. Using this strategy, mismatches between the protospacer and theunedited allele should disfavor nicking by the sgRNA until after theediting event on the PAM strand takes place.

Trans Prime Editing

As used herein, the term “trans prime editing” refers to a modified formof prime editing that utilizes a split PEgRNA, i.e., wherein the PEgRNAis separated into two separate molecules: an sgRNA and a trans primeediting RNA template (tPERT). The sgRNA serves to target the primeeditor (or more generally, to target the napDNAbp component of the primeeditor) to the desired genomic target site, while the tPERT is used bythe polymerase (e.g., a reverse transcriptase) to write new DNA sequenceinto the target locus once the tPERT is recruited in trans to the primeeditor by the interaction of binding domains located on the prime editorand on the tPERT. In one embodiment, the binding domains can includeRNA-protein recruitment moieties, such as a MS2 aptamer located on thetPERT and an MS2cp protein fused to the prime editor. An advantage oftrans prime editing is that by separating the DNA synthesis templatefrom the guide RNA, one can potentially use longer length templates.

An embodiment of trans prime editing is shown in FIGS. 3G and 3H. FIG.3G shows the composition of the trans prime editor complex on the left(“RP-PE:gRNA complex), which comprises an napDNAbp fused to each of apolymerase (e.g., a reverse transcriptase) and a rPERT recruitingprotein (e.g., MS2sc), and which is complexed with a guide RNA. FIG. 3Gfurther shows a separate tPERT molecule, which comprises the extensionarm features of a PEgRNA, including the DNA synthesis template and theprimer binding sequence. The tPERT molecule also includes an RNA-proteinrecruitment domain (which, in this case, is a stem loop structure andcan be, for example, MS2 aptamer). As depicted in the process describedin FIG. 3H, the RP-PE:gRNA complex binds to and nicks the target DNAsequence. Then, the recruiting protein (RP) recruits a tPERT toco-localize to the prime editor complex bound to the DNA target site,thereby allowing the primer binding site to bind to the primer sequenceon the nicked strand, and subsequently, allowing the polymerase (e.g.,RT) to synthesize a single strand of DNA against the DNA synthesistemplate up through the 5′ of the tPERT.

While the tPERT is shown in FIG. 3G and FIG. 3H as comprising the PBSand DNA synthesis template on the 5′ end of the RNA-protein recruitmentdomain, the tPERT in other configurations may be designed with the PBSand DNA synthesis template located on the 3′ end of the RNA-proteinrecruitment domain. However, the tPERT with the 5′ extension has theadvantage that synthesis of the single strand of DNA will naturallyterminate at the 5′ end of the tPERT and thus, does not risk using anyportion of the RNA-protein recruitment domain as a template during theDNA synthesis stage of prime editing.

Trans Prime Editor RNA Template (tPERT)

As used herein, a “trans prime editor RNA template (tPERT)” refers to acomponent used in trans prime editing, a modified version of primeediting which operates by separating the PEgRNA into two distinctmolecules: a guide RNA and a tPERT molecule. The tPERT molecule isprogrammed to co-localize with the prime editor complex at a target DNAsite, bringing the primer binding site and the DNA synthesis template tothe prime editor in trans. For example, see FIG. 3G for an embodiment ofa trans prime editor (tPE) which shows a two-component system comprising(1) an RP-PE:gRNA complex and (2) a tPERT that includes the primerbinding site and the DNA synthesis template joined to an RNA-proteinrecruitment domain, wherein the RP (recruiting protein) component of theRP-PE:gRNA complex recruits the tPERT to a target site to be edited,thereby associating the PBS and DNA synthesis template with the primeeditor in trans. Said another way, the tPERT is engineered to contain(all or part of) the extension arm of a PEgRNA, which includes theprimer binding site and the DNA synthesis template.

Transitions

As used herein, “transitions” refer to the interchange of purinenucleobases (A↔G) or the interchange of pyrimidine nucleobases (C↔T).This class of interchanges involves nucleobases of similar shape. Thecompositions and methods disclosed herein are capable of inducing one ormore transitions in a target DNA molecule. The compositions and methodsdisclosed herein are also capable of inducing both transitions andtransversion in the same target DNA molecule. These changes involve A↔G,G↔A, C↔T, or T↔C. In the context of a double-strand DNA withWatson-Crick paired nucleobases, transversions refer to the followingbase pair exchanges: A:T↔G:C, G:G↔A:T, C:G↔T:A, or T:A↔C:G. Thecompositions and methods disclosed herein are capable of inducing one ormore transitions in a target DNA molecule. The compositions and methodsdisclosed herein are also capable of inducing both transitions andtransversion in the same target DNA molecule, as well as othernucleotide changes, including deletions and insertions.

Transversions

As used herein, “transversions” refer to the interchange of purinenucleobases for pyrimidine nucleobases, or in the reverse and thus,involve the interchange of nucleobases with dissimilar shape. Thesechanges involve T↔A, T↔G, C↔G, C↔A, A↔T, A↔C, G↔C, and G↔T. In thecontext of a double-strand DNA with Watson-Crick paired nucleobases,transversions refer to the following base pair exchanges: T:A↔A:T,T:A↔G:C, C:G↔G:C, C:G↔A:T, A:T↔T:A, A:T↔C:G, G:C↔C:G, and G:C↔T:A. Thecompositions and methods disclosed herein are capable of inducing one ormore transversions in a target DNA molecule. The compositions andmethods disclosed herein are also capable of inducing both transitionsand transversion in the same target DNA molecule, as well as othernucleotide changes, including deletions and insertions.

Treatment

The terms “treatment,” “treat,” and “treating,” refer to a clinicalintervention aimed to reverse, alleviate, delay the onset of, or inhibitthe progress of a disease or disorder, or one or more symptoms thereof,as described herein. As used herein, the terms “treatment,” “treat,” and“treating” refer to a clinical intervention aimed to reverse, alleviate,delay the onset of, or inhibit the progress of a disease or disorder, orone or more symptoms thereof, as described herein. In some embodiments,treatment may be administered after one or more symptoms have developedand/or after a disease has been diagnosed. In other embodiments,treatment may be administered in the absence of symptoms, e.g., toprevent or delay onset of a symptom or inhibit onset or progression of adisease. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors).Treatment may also be continued after symptoms have resolved, forexample, to prevent or delay their recurrence.

Trinucleotide Repeat Disorder

As used herein, a “trinucleotide repeat disorder” (or alternatively,“expansion repeat disorder” or “repeat expansion disorder”) refers to aset of genetic disorders which are cause by “trinucleotide repeatexpansion,” which is a kind of mutation where a certain trinucleotiderepeats in certain genes or introns. Trinucleotide repeats were oncethought to be commonplace iterations in the genome, but the 1990sclarified these disorders. These apparently ‘benign’ stretches of DNAcan sometimes expand and cause disease. Several defining features areshared amongst disorders caused by trinucleotide repeat expansions.First, the mutant repeats show both somatic and germline instabilityand, more frequently, they expand rather than contract in successivetransmissions. Secondly, an earlier age of onset and increasing severityof phenotype in subsequent generations (anticipation) generally arecorrelated with larger repeat length. Finally, the parental origin ofthe disease allele can often influence anticipation, with paternaltransmissions carrying a greater risk of expansion for many of thesedisorders.

Triplet expansion is thought to be caused by slippage during DNAreplication. Due to the repetitive nature of the DNA sequence in theseregions ‘loop out’ structures may form during DNA replication whilemaintaining complementary base pairing between the parent strand anddaughter strand being synthesized. If the loop out structure is formedfrom sequence on the daughter strand this will result in an increase inthe number of repeats. However, if the loop out structure is formed onthe parent strand a decrease in the number of repeats occurs. It appearsthat expansion of these repeats is more common than reduction. Generallythe larger the expansion the more likely they are to cause disease orincrease the severity of disease. This property results in thecharacteristic of anticipation seen in trinucleotide repeat disorders.Anticipation describes the tendency of age of onset to decrease andseverity of symptoms to increase through successive generations of anaffected family due to the expansion of these repeats.

Nucleotide repeat disorders may include those in which the tripletrepeat occurs in a non-coding region (i.e., a non-coding trinucleotiderepeat disorder) or in a coding region

The prime editor (PE) system described herein may use to treatnucleotide repeat disorders, which may include fragile X syndrome(FRAXA), fragile XE MR (FRAXE), Freidreich ataxia (FRDA), myotonicdystrophy (DM), spinocerebellar ataxia type 8 (SCA8), andspinocerebellar ataxia type 12 (SCA12), among others.

Upstream

As used herein, the terms “upstream” and “downstream” are terms ofrelativity that define the linear position of at least two elementslocated in a nucleic acid molecule (whether single or double-stranded)that is orientated in a 5′-to-3′ direction. In particular, a firstelement is upstream of a second element in a nucleic acid molecule wherethe first element is positioned somewhere that is 5′ to the secondelement. For example, a SNP is upstream of a Cas9-induced nick site ifthe SNP is on the 5′ side of the nick site. Conversely, a first elementis downstream of a second element in a nucleic acid molecule where thefirst element is positioned somewhere that is 3′ to the second element.For example, a SNP is downstream of a Cas9-induced nick site if the SNPis on the 3′ side of the nick site. The nucleic acid molecule can be aDNA (double or single stranded). RNA (double or single stranded), or ahybrid of DNA and RNA. The analysis is the same for single strandnucleic acid molecule and a double strand molecule since the termsupstream and downstream are in reference to only a single strand of anucleic acid molecule, except that one needs to select which strand ofthe double stranded molecule is being considered. Often, the strand of adouble stranded DNA which can be used to determine the positionalrelativity of at least two elements is the “sense” or “coding” strand.In genetics, a “sense” strand is the segment within double-stranded DNAthat runs from 5′ to 3′, and which is complementary to the antisensestrand of DNA, or template strand, which runs from 3′ to 5′. Thus, as anexample, a SNP nucleobase is “downstream” of a promoter sequence in agenomic DNA (which is double-stranded) if the SNP nucleobase is on the3′ side of the promoter on the sense or coding strand.

Variant

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature, e.g., a variant Cas9 is a Cas9 comprising one or more changes inamino acid residues as compared to a wild type Cas9 amino acid sequence.The term “variant” encompasses homologous proteins having at least 75%,or at least 80%, or at least 85%, or at least 90%, or at least 95%, orat least 99% percent identity with a reference sequence and having thesame or substantially the same functional activity or activities as thereference sequence. The term also encompasses mutants, truncations, ordomains of a reference sequence, and which display the same orsubstantially the same functional activity or activities as thereference sequence.

Vector

The term “vector,” as used herein, refers to a nucleic acid that can bemodified to encode a gene of interest and that is able to enter into ahost cell, mutate and replicate within the host cell, and then transfera replicated form of the vector into another host cell. Exemplarysuitable vectors include viral vectors, such as retroviral vectors orbacteriophages and filamentous phage, and conjugative plasmids.Additional suitable vectors will be apparent to those of skill in theart based on the instant disclosure.

Wild Type

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms.

5′ Endogenous DNA Flap

As used herein, the term “5′ endogenous DNA flap” refers to the strandof DNA situated immediately downstream of the PE-induced nick site inthe target DNA. The nicking of the target DNA strand by PE exposes a 3′hydroxyl group on the upstream side of the nick site and a 5′ hydroxylgroup on the downstream side of the nick site. The endogenous strandending in the 3′ hydroxyl group is used to prime the DNA polymerase ofthe prime editor (e.g., wherein the DNA polymerase is a reversetranscriptase). The endogenous strand on the downstream side of the nicksite and which begins with the exposed 5′ hydroxyl group is referred toas the “5′ endogenous DNA flap” and is ultimately removed and replacedby the newly synthesized replacement strand (i.e., “3′ replacement DNAflap”) the encoded by the extension of the PEgRNA.

5′ Endogenous DNA Flap Removal

As used herein, the term “5′ endogenous DNA flap removal” or “5′ flapremoval” refers to the removal of the 5′ endogenous DNA flap that formswhen the RT-synthesized single-strand DNA flap competitively invades andhybridizes to the endogenous DNA, displacing the endogenous strand inthe process. Removing this endogenous displaced strand can drive thereaction towards the formation of the desired product comprising thedesired nucleotide change. The cell's own DNA repair enzymes maycatalyze the removal or excision of the 5′ endogenous flap (e.g., a flapendonuclease, such as EXO1 or FEN1). Also, host cells may be transformedto express one or more enzymes that catalyze the removal of said 5′endogenous flaps, thereby driving the process toward product formation(e.g., a flap endonuclease). Flap endonucleases are known in the art andcan be found described in Patel et al., “Flap endonucleases pass5′-flaps through a flexible arch using a disorder-thread-order mechanismto confer specificity for free 5′-ends,” Nucleic Acids Research, 2012,40(10): 4507-4519 and Tsutakawa et al., “Human flap endonucleasestructures, DNA double-base flipping, and a unified understanding of theFEN1 superfamily,” Cell, 2011, 145(2): 198-211 (each of which areincorporated herein by reference).

3′ replacement DNA Flap

As used herein, the term “3′ replacement DNA flap” or simply,“replacement DNA flap,” refers to the strand of DNA that is synthesizedby the prime editor and which is encoded by the extension arm of theprime editor PEgRNA. More in particular, the 3′ replacement DNA flap isencoded by the polymerase template of the PEgRNA. The 3′ replacement DNAflap comprises the same sequence as the 5′ endogenous DNA flap exceptthat it also contains the edited sequence (e.g., single nucleotidechange). The 3′ replacement DNA flap anneals to the target DNA,displacing or replacing the 5′ endogenous DNA flap (which can beexcised, for example, by a 5′ flap endonuclease, such as FEN1 or EXO1)and then is ligated to join the 3′ end of the 3′ replacement DNA flap tothe exposed 5′ hydroxyl end of endogenous DNA (exposed after excision ofthe 5′ endogenous DNA flap, thereby reforming a phosphodiester bond andinstalling the 3′ replacement DNA flap to form a heteroduplex DNAcontaining one edited strand and one unedited strand. DNA repairprocesses resolve the heteroduplex by copying the information in theedited strand to the complementary strand permanently installs the editin to the DNA. This resolution process can be driven further tocompletion by nicking the unedited strand, i.e., by way of“second-strand nicking,” as described herein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Adoption of the clustered regularly interspaced short palindromic repeat(CRISPR) system for genome editing has revolutionized the lifesciences¹⁻³. Although gene disruption using CRISPR is now routine, theprecise installation of single nucleotide edits remains a majorchallenge, despite being necessary for studying or correcting a largenumber of disease-causative mutations. Homology directed repair (HDR) iscapable of achieving such edits, but suffers from low efficiency (often<5%), a requirement for donor DNA repair templates, and deleteriouseffects of double-stranded DNA break (DSB) formation. Recently, Prof.David Liu et al.'s laboratory developed base editing, which achievesefficient single nucleotide editing without DSBs. Base editors (BEs)combine the CRISPR system with base-modifying deaminase enzymes toconvert target C·G or A·T base pairs to A·T or G·C, respectively⁴⁻⁶.Although already widely used by researchers worldwide, current BEsenable only four of the twelve possible base pair conversions and areunable to correct small insertions or deletions. Moreover, the targetingscope of base editing is limited by the editing of non-target C or Abases adjacent to the target base (“bystander editing”) and by therequirement that a PAM sequence exist 15±2 bp from the target base.Overcoming these limitations would therefore greatly broaden the basicresearch and therapeutic applications of genome editing.

The present disclosure proposes a new precision editing approach thatoffers many of the benefits of base editing—namely, avoidance of doublestrand breaks and donor DNA repair templates—while overcoming its majorlimitations. The proposed approach described herein achieves the directinstallation of edited DNA strands at target genomic sites usingtarget-primed reverse transcription (TPRT). In the design discussedherein, CRISPR guide RNA (gRNA) will be engineered to carry a reversetranscriptase (RT) template sequence encoding a single-stranded DNAcomprising a desired nucleotide change. The CRISPR nuclease(Cas9)-nicked target site DNA will serve as the primer for reversetranscription of the template sequence on the modified gRNA, allowingfor direct incorporation of any desired nucleotide edit.

Accordingly, the present invention relates in part to the discovery thatthe mechanism of target-primed reverse transcription (TPRT) can beleveraged or adapted for conducting precision CRISPR/Cas-based genomeediting with high efficiency and genetic flexibility (e.g., as depictedin various embodiments of FIGS. 1A-1F). The inventors have proposedherein to use Cas protein-reverse transcriptase fusions to target aspecific DNA sequence with a modified guide RNA (“an extended guideRNA”), generate a single strand nick at the target site, and use thenicked DNA as a primer for reverse transcription of an engineeredreverse transcriptase template that is integrated into the extendedguide RNA. The newly synthesized strand would be homologous to thegenomic target sequence except for the inclusion of a desired nucleotidechange (e.g., a single nucleotide change, a deletion, or an insertion,or a combination thereof). The newly synthesize strand of DNA may bereferred to as a single strand DNA flap, which would compete forhybridization with the complementary homologous endogenous DNA strand,thereby displacing the corresponding endogenous strand. Resolution ofthis hybridized intermediate can include removal of the resultingdisplaced flap of endogenous DNA (e.g., with a 5′ end DNA flapendonuclease, FEN1), ligation of the synthesized single strand DNA flapto the target DNA, and assimilation of the desired nucleotide change asa result of cellular DNA repair and/or replication processes. Becausetemplated DNA synthesis offers single nucleotide precision, the scope ofthis approach is very broad and could foreseeably be used for myriadapplications in basic science and therapeutics.

[1] napDNAbp

The prime editors and trans prime editors described herein may comprisea nucleic acid programmable DNA binding protein (napDNAbp).

In one aspect, a napDNAbp can be associated with or complexed with atleast one guide nucleic acid (e.g., guide RNA or a PEgRNA), whichlocalizes the napDNAbp to a DNA sequence that comprises a DNA strand(i.e., a target strand) that is complementary to the guide nucleic acid,or a portion thereof (e.g., the spacer of a guide RNA which anneals tothe protospacer of the DNA target). In other words, the guidenucleic-acid “programs” the napDNAbp (e.g., Cas9 or equivalent) tolocalize and bind to complementary sequence of the protospacer in theDNA.

Any suitable napDNAbp may be used in the prime editors described herein.In various embodiments, the napDNAbp may be any Class 2 CRISPR-Cassystem, including any type II, type V or type VI CRISPR-Cas enzyme.Given the rapid development of CRISPR-Cas as a tool for genome editing,there have been constant developments in the nomenclature used todescribe and/or identify CRISPR-Cas enzymes, such as Cas9 and Cas9orthologs. This application references CRISPR-Cas enzymes withnomenclature that may be old and/or new. The skilled person will be ableto identify the specific CRISPR-Cas enzyme being referenced in thisApplication based on the nomenclature that is used, whether it is old(i.e., “legacy”) or new nomenclature. CRISPR-Cas nomenclature isextensively discussed in Makarova et al., “Classification andNomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPRJournal, Vol. 1. No. 5, 2018, the entire contents of which areincorporated herein by reference. The particular CRISPR-Cas nomenclatureused in any given instance in this application is not limiting in anyway and the skilled person will be able to identify which CRISPR-Casenzyme is being referenced.

For example, the following type II, type V, and type VI Class 2CRISPR-Cas enzymes have the following art-recognized old (i.e., legacy)and new names. Each of these enzymes, and/or variants thereof, may beused with the prime editors described herein:

Legacy nomenclature Current nomenclature* type II CRISPR-Cas enzymesCas9 same type V CRISPR-Cas enzymes Cpf1 Cas12a CasX Cas12e C2c1 Cas12b1Cas12b2 same C2c3 Cas12c CasY Cas12d C2c4 same C2c8 same C2c5 same C2c10same C2c9 same type VI CRISPR-Cas enzymes C2c2 Cas13a Cas13d same C2c7Cas13c C2c6 Cas13b *See Makarova et al., The CRISPR Journal, Vol. 1, No.5, 2018

Without being bound by theory, the mechanism of action of certainnapDNAbp contemplated herein includes the step of forming an R-loopwhereby the napDNAbp induces the unwinding of a double-strand DNAtarget, thereby separating the strands in the region bound by thenapDNAbp. The guide RNA spacer then hybridizes to the “target strand” atthe protospacer sequence. This displaces a “non-target strand” that iscomplementary to the target strand, which forms the single strand regionof the R-loop. In some embodiments, the napDNAbp includes one or morenuclease activities, which then cut the DNA leaving various types oflesions. For example, the napDNAbp may comprises a nuclease activitythat cuts the non-target strand at a first location, and/or cuts thetarget strand at a second location. Depending on the nuclease activity,the target DNA can be cut to form a “double-stranded break” whereby bothstrands are cut. In other embodiments, the target DNA can be cut at onlya single site, i.e., the DNA is “nicked” on one strand. ExemplarynapDNAbp with different nuclease activities include “Cas9 nickase”(“nCas9”) and a deactivated Cas9 having no nuclease activities (“deadCas9” or “dCas9”).

The below description of various napDNAbps which can be used inconnection with the presently disclose prime editors is not meant to belimiting in any way. The prime editors may comprise the canonicalSpCas9, or any ortholog Cas9 protein, or any variant Cas9protein—including any naturally occurring variant, mutant, or otherwiseengineered version of Cas9—that is known or which can be made or evolvedthrough a directed evolutionary or otherwise mutagenic process. Invarious embodiments, the Cas9 or Cas9 variants have a nickase activity,i.e., only cleave of strand of the target DNA sequence. In otherembodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e.,are “dead” Cas9 proteins. Other variant Cas9 proteins that may be usedare those having a smaller molecular weight than the canonical SpCas9(e.g., for easier delivery) or having modified or rearranged primaryamino acid structure (e.g., the circular permutant formats).

The prime editors described herein may also comprise Cas9 equivalents,including Cas12a (Cpf1) and Cas12b1 proteins which are the result ofconvergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9variant, or Cas9 equivalents) may also may also contain variousmodifications that alter/enhance their PAM specifities. Lastly, theapplication contemplates any Cas9, Cas9 variant, or Cas9 equivalentwhich has at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atleast 99.9% sequence identity to a reference Cas9 sequence, such as areferences SpCas9 canonical sequence or a reference Cas9 equivalent(e.g., Cas12a (Cpf1)).

The napDNAbp can be a CRISPR (clustered regularly interspaced shortpalindromic repeat)-associated nuclease. As outlined above, CRISPR is anadaptive immune system that provides protection against mobile geneticelements (viruses, transposable elements and conjugative plasmids).CRISPR clusters contain spacers, sequences complementary to antecedentmobile elements, and target invading nucleic acids. CRISPR clusters aretranscribed and processed into CRISPR RNA (crRNA). In type II CRISPRsystems correct processing of pre-crRNA requires a trans-encoded smallRNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. ThetracrRNA serves as a guide for ribonuclease 3-aided processing ofpre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaveslinear or circular dsDNA target complementary to the spacer. The targetstrand not complementary to crRNA is first cut endonucleolytically, thentrimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavagetypically requires protein and both RNAs. However, single guide RNAs(“sgRNA”, or simply “gRNA”) can be engineered so as to incorporateaspects of both the crRNA and tracrRNA into a single RNA species. See,e.g., Jinek M. et al., Science 337:816-821(2012), the entire contents ofwhich is hereby incorporated by reference.

In some embodiments, the napDNAbp directs cleavage of one or bothstrands at the location of a target sequence, such as within the targetsequence and/or within the complement of the target sequence. In someembodiments, the napDNAbp directs cleavage of one or both strands withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, ormore base pairs from the first or last nucleotide of a target sequence.In some embodiments, a vector encodes a napDNAbp that is mutated to withrespect to a corresponding wild-type enzyme such that the mutatednapDNAbp lacks the ability to cleave one or both strands of a targetpolynucleotide containing a target sequence. For example, anaspartate-to-alanine substitution (D10A) in the RuvC I catalytic domainof Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves bothstrands to a nickase (cleaves a single strand). Other examples ofmutations that render Cas9 a nickase include, without limitation, H840A,N854A, and N863A in reference to the canonical SpCas9 sequence, or toequivalent amino acid positions in other Cas9 variants or Cas9equivalents.

As used herein, the term “Cas protein” refers to a full-length Casprotein obtained from nature, a recombinant Cas protein having asequences that differs from a naturally occurring Cas protein, or anyfragment of a Cas protein that nevertheless retains all or a significantamount of the requisite basic functions needed for the disclosedmethods, i.e., (i) possession of nucleic-acid programmable binding ofthe Cas protein to a target DNA, and (ii) ability to nick the target DNAsequence on one strand. The Cas proteins contemplated herein embraceCRISPR Cas 9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9nickase (nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs,or paralogs, whether naturally occurring or non-naturally occurring(e.g., engineered or recombinant), and may include a Cas9 equivalentfrom anyClass 2 CRISPR system (e.g., type II, V, VI), including Cas12a(Cpf1), Cas12e (CasX), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), C2c4,C2c8, C2c5, C2c10, C2c9 Cas13a (C2c2), Cas13d, Cas13c (C2c7), Cas13b(C2c6), and Cas13b. Further Cas-equivalents are described in Makarova etal., “C2c2 is a single-component programmable RNA-guided RNA-targetingCRISPR effector,” Science 2016; 353(6299) and Makarova et al.,“Classification and Nomenclature of CRISPR-Cas Systems: Where fromHere?,” The CRISPR Journal, Vol. 1. No. 5, 2018, the contents of whichare incorporated herein by reference.

The terms “Cas9” or “Cas9 nuclease” or “Cas9 moiety” or “Cas9 domain”embrace any naturally occurring Cas9 from any organism, anynaturally-occurring Cas9 equivalent or functional fragment thereof, anyCas9 homolog, ortholog, or paralog from any organism, and any mutant orvariant of a Cas9, naturally-occurring or engineered. The term Cas9 isnot meant to be particularly limiting and may be referred to as a “Cas9or equivalent.” Exemplary Cas9 proteins are further described hereinand/or are described in the art and are incorporated herein byreference. The present disclosure is unlimited with regard to theparticular Cas9 that is employed in the prime editor (PE) of theinvention.

As noted herein, Cas9 nuclease sequences and structures are well knownto those of skill in the art (see, e.g., “Complete genome sequence of anM1 strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W.M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S.,Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G.,Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W.,Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNAand host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M.,Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., CharpentierE., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K.,Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science337:816-821(2012), the entire contents of each of which are incorporatedherein by reference).

Examples of Cas9 and Cas9 equivalents are provided as follows; however,these specific examples are not meant to be limiting. The primer editorof the present disclosure may use any suitable napDNAbp, including anysuitable Cas9 or Cas9 equivalent.

A. Wild Type Canonical SpCas9

In one embodiment, the primer editor constructs described herein maycomprise the “canonical SpCas9” nuclease from S. pyogenes, which hasbeen widely used as a tool for genome engineering and is categorized asthe type II subgroup of enzymes of the Class 2 CRISPR-Cas systems. ThisCas9 protein is a large, multi-domain protein containing two distinctnuclease domains. Point mutations can be introduced into Cas9 to abolishone or both nuclease activities, resulting in a nickase Cas9 (nCas9) ordead Cas9 (dCas9), respectively, that still retains its ability to bindDNA in a sgRNA-programmed manner. In principle, when fused to anotherprotein or domain, Cas9 or variant thereof (e.g., nCas9) can target thatprotein to virtually any DNA sequence simply by co-expression with anappropriate sgRNA. As used herein, the canonical SpCas9 protein refersto the wild type protein from Streptococcus pyogenes having thefollowing amino acid sequence:

Description Sequence SEQ ID NO: SpCas9MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN SEQ ID NO: 18 Strepto-TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR coccusKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH pyogenesERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL M1RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ SwissProtTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL AccessionPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS No. Q99ZW2KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI Wild typeLRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SpCas9ATGGATAAAAAATATAGCATTGGCCTGGATATTGGC SEQ ID NO: 19 ReverseACCAACAGCGTGGGCTGGGCGGTGATTACCGATGAA translationTATAAAGTGCCGAGCAAAAAATTTAAAGTGCTGGGC ofAACACCGATCGCCATAGCATTAAAAAAAACCTGATT SwissProtGGCGCGCTGCTGTTTGATAGCGGCGAAACCGCGGAA AccessionGCGACCCGCCTGAAACGCACCGCGCGCCGCCGCTAT No. Q99ZW2ACCCGCCGCAAAAACCGCATTTGCTATCTGCAGGAA Strepto-ATTTTTAGCAACGAAATGGCGAAAGTGGATGATAGC coccusTTTTTTCATCGCCTGGAAGAAAGCTTTCTGGTGGAAG pyogenesAAGATAAAAAACATGAACGCCATCCGATTTTTGGCAACATTGTGGATGAAGTGGCGTATCATGAAAAATATCCGACCATTTATCATCTGCGCAAAAAACTGGTGGATAGCACCGATAAAGCGGATCTGCGCCTGATTTATCTGGCGCTGGCGCATATGATTAAATTTCGCGGCCATTTTCTGATTGAAGGCGATCTGAACCCGGATAACAGCGATGTGGATAAACTGTTTATTCAGCTGGTGCAGACCTATAACCAGCTGTTTGAAGAAAACCCGATTAACGCGAGCGGCGTGGATGCGAAAGCGATTCTGAGCGCGCGCCTGAGCAAAAGCCGCCGCCTGGAAAACCTGATTGCGCAGCTGCCGGGCGAAAAAAAAAACGGCCTGTTTGGCAACCTGATTGCGCTGAGCCTGGGCCTGACCCCGAACTTTAAAAGCAACTTTGATCTGGCGGAAGATGCGAAACTGCAGCTGAGCAAAGATACCTATGATGATGATCTGGATAACCTGCTGGCGCAGATTGGCGATCAGTATGCGGATCTGTTTCTGGCGGCGAAAAACCTGAGCGATGCGATTCTGCTGAGCGATATTCTGCGCGTGAACACCGAAATTACCAAAGCGCCGCTGAGCGCGAGCATGATTAAACGCTATGATGAACATCATCAGGATCTGACCCTGCTGAAAGC GCTGGTGCGCCAGCAGCTGCCGGAAAAATATAAAGAAATTTTTTTTGATCAGAGCAAAAACGGCTATGCGGGCTATATTGATGGCGGCGCGAGCCAGGAAGAATTTTATAAATTTATTAAACCGATTCTGGAAAAAATGGATG GCACCGAAGAACTGCTGGTGAAACTGAACCGCGAAGATCTGCTGCGCAAACAGCGCACCTTTGATAACGGCAGCATTCCGCATCAGATTCATCTGGGCGAACTGCATGCGATTCTGCGCCGCCAGGAAGATTTTTATCCGTTTCTGAAAGATAACCGCGAAAAAATTGAAAAAATTCTGACCTTTCGCATTCCGTATTATGTGGGCCCGCTGGCGCGCGGCAACAGCCGCTTTGCGTGGATGACCCGCAAAAGCGAAGAAACCATTACCCCGTGGAACTTTGAAGAAGTGGTGGATAAAGGCGCGAGCGCGCAGAGCTTTATTGAACGCATGACCAACTTTGATAAAAACCTGCCGAACGAAAAAGTGCTGCCGAAACATAGCCTGCTGTATGAATATTTTACCGTGTATAACGAACTGACCAAAGTGAAATATGTGACCGAAGGCATGCGCAAACCGGCGTTTCTGAGCGGCGAACAGAAAAAAGCGATTGTGGATCTGCTGTTTAAAACCAACCGCAAAGTGACCGTGAAACAGCTGAAAGAAGATTATTTTAAAAAAATTGAATGCTTTGATAGCGTGGAAATTAGCGGCGTGGAAGATCGCTTTAACGCGAGCCTGGGCACCTATCATGATCTGCTGAAAATTATTAAAGATAAAGATTTTCTGGATAACGAAGAAAACGAAGATATTCTGGAAGATATTGTGCTGACCCTGACCCTGTTTGAAGATCGCGAAATGATTGAAGAACGCCTGAAAACCTATGCGCATCTGTTTGATGATAAAGTGATGAAACAGCTGAAACGCCGCCGCTATACCGGCTGGGGCCGCCTGAGCCGCAAACTGATTAACGGCATTCGCGATAAACAGAGCGGCAAAACCATTCTGGATTTTCTGAAAAGCGATGGCTTTGCGAACCGCAACTTTATGCAGCTGATTCATGATGATAGCCTGACCTTTAAAGAAGATATTC AGAAAGCGCAGGTGAGCGGCCAGGGCGATAGCCTGCATGAACATATTGCGAACCTGGCGGGCAGCCCGGCGATTAAAAAAGGCATTCTGCAGACCGTGAAAGTGGTGGATGAACTGGTGAAAGTGATGGGCCGCCATAAACCG GAAAACATTGTGATTGAAATGGCGCGCGAAAACCAGACCACCCAGAAAGGCCAGAAAAACAGCCGCGAAC GCATGAAACGCATTGAAGAAGGCATTAAAGAACTGGGCAGCCAGATTCTGAAAGAACATCCGGTGGAAAA CACCCAGCTGCAGAACGAAAAACTGTATCTGTATTATCTGCAGAACGGCCGCGATATGTATGTGGATCAGGAACTGGATATTAACCGCCTGAGCGATTATGATGTGGATCATATTGTGCCGCAGAGCTTTCTGAAAGATGATAGCATTGATAACAAAGTGCTGACCCGCAGCGATAAAAA CCGCGGCAAAAGCGATAACGTGCCGAGCGAAGAAGTGGTGAAAAAAATGAAAAACTATTGGCGCCAGCTGCTGAACGCGAAACTGATTACCCAGCGCAAATTTGATA ACCTGACCAAAGCGGAACGCGGCGGCCTGAGCGAACTGGATAAAGCGGGCTTTATTAAACGCCAGCTGGTGGAAACCCGCCAGATTACCAAACATGTGGCGCAGATTCTGGATAGCCGCATGAACACCAAATATGATGAAAACGATAAACTGATTCGCGAAGTGAAAGTGATTACCCTGAAAAGCAAACTGGTGAGCGATTTTCGCAAAGATTTTCAGTTTTATAAAGTGCGCGAAATTAACAACTATCATCATGCGCATGATGCGTATCTGAACGCGGTGGTGGGCACCGCGCTGATTAAAAAATATCCGAAACTGGAAAGCGAATTTGTGTATGGCGATTATAAAGTGTATGATGTG CGCAAAATGATTGCGAAAAGCGAACAGGAAATTGGCAAAGCGACCGCGAAATATTTTTTTTATAGCAACATTATGAACTTTTTTAAAACCGAAATTACCCTGGCGAACGGCGAAATTCGCAAACGCCCGCTGATTGAAACCAA CGGCGAAACCGGCGAAATTGTGTGGGATAAAGGCCGCGATTTTGCGACCGTGCGCAAAGTGCTGAGCATGC CGCAGGTGAACATTGTGAAAAAAACCGAAGTGCAGACCGGCGGCTTTAGCAAAGAAAGCATTCTGCCGAAA CGCAACAGCGATAAACTGATTGCGCGCAAAAAAGATTGGGATCCGAAAAAATATGGCGGCTTTGATAGCCCGACCGTGGCGTATAGCGTGCTGGTGGTGGCGAAAGT GGAAAAAGGCAAAAGCAAAAAACTGAAAAGCGTGAAAGAACTGCTGGGCATTACCATTATGGAACGCAGCAGCTTTGAAAAAAACCCGATTGATTTTCTGGAAGCGAAAGGCTATAAAGAAGTGAAAAAAGATCTGATTATTAAACTGCCGAAATATAGCCTGTTTGAACTGGAAAACG GCCGCAAACGCATGCTGGCGAGCGCGGGCGAACTGCAGAAAGGCAACGAACTGGCGCTGCCGAGCAAATA TGTGAACTTTCTGTATCTGGCGAGCCATTATGAAAAACTGAAAGGCAGCCCGGAAGATAACGAACAGAAAC AGCTGTTTGTGGAACAGCATAAACATTATCTGGATGAAATTATTGAACAGATTAGCGAATTTAGCAAACGCGTGATTCTGGCGGATGCGAACCTGGATAAAGTGCTGAGCGCGTATAACAAACATCGCGATAAACCGATTCGCGAACAGGCGGAAAACATTATTCATCTGTTTACCCTGACCAACCTGGGCGCGCCGGCGGCGTTTAAATATTTTGATACCACCATTGATCGCAAACGCTATACCAGCACCAAAGAAGTGCTGGATGCGACCCTGATTCATCAGAGCATTACCGGCCTGTATGAAACCCGCATTGATCTGAGCC AGCTGGGCGGCGAT

The prime editors described herein may include canonical SpCas9, or anyvariant thereof having at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% sequence identity with a wild type Cas9sequence provided above. These variants may include SpCas9 variantscontaining one or more mutations, including any known mutation reportedwith the SwissProt Accession No. Q99ZW2 (SEQ ID NO: 18) entry, whichinclude:

SpCas9 mutation (relative to the amino Function/Characteristic (asreported) acid sequence of (see UniProtKB - Q99ZW2 the canonical SpCas9(CAS9_STRPT1) entry- sequence, SEQ ID NO: 18) incorporated herein byreference) D10A Nickase mutant which cleaves the protospacer strand (butno cleavage of non-protospacer strand) S15A Decreased DNA cleavageactivity R66A Decreased DNA cleavage activity R70A No DNA cleavage R74ADecreased DNA cleavage R78A Decreased DNA cleavage 97-150 deletion Nonuclease activity R165A Decreased DNA cleavage 175-307 deletion About50% decreased DNA cleavage 312-409 deletion No nuclease activity E762ANickase H840A Nickase mutant which cleaves the non-protospacer strandbut does not cleave the protospacer strand N854A Nickase N863A NickaseH982A Decreased DNA cleavage D986A Nickase 1099-1368 deletion Nonuclease activity R1333A Reduced DNA binding

Other wild type SpCas9 sequences that may be used in the presentdisclosure, include:

Description Sequence SEQ ID NO: SpCas9ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCA SEQ ID NO: 20 Strepto-CAAATAGCGTCGGATGGGCGGTGATCACTGATGATTA coccusTAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAAT pyogenesACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGG MGAS1882CTCTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGAC wild typeTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGT NC_017053.1CGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGC TAGGAGGTGACTGA SpCas9MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNT SEQ ID NO: 21 Strepto-DRHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKN coccusRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP pyogenesIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLA MGAS1882LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFE wild typeENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGL NC_017053.1FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDSpCas9 ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCAC SEQ ID NO: 22 Strepto-TAATTCCGTTGGATGGGCTGTCATAACCGATGAATACA coccusAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACAC pyogenesAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCC wild typeTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCG SWBC2D7W014CCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTA CAAGGATGACGATGACAAGGCTGCAGGASpCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO: 23 Strepto-RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI coccusCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF pyogenesGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL wild typeAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE EncodedNPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF product ofGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN SWBC2D7W014LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKV SSDYKDHDGDYKDHDIDYKDDDDKAAGSpCas9 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCA SEQ ID NO: 24 Strepto-CAAATAGCGTCGGATGGGCGGTGATCACTGATGAATA coccusTAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAAT pyogenesACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGG M1GAS wildCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGAC typeTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGT NC_002737.2CGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAG GAGGTGACTGA SpCas9MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO: 25 Strepto-RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI coccusCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF pyogenesGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL M1GAS wildAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE typeNPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF EncodedGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN product ofLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL NC_002737.2SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN (100%GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE identicalDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN to theREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW canonicalNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY Q99ZW2EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK wild type)TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT STKEVLDATLIHQSITGLYETRIDLSQLGGD

The prime editors described herein may include any of the above SpCas9sequences, or any variant thereof having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto.

B. Wild Type Cas9 Orthologs

In other embodiments, the Cas9 protein can be a wild type Cas9 orthologfrom another bacterial species different from the canonical Cas9 from S.pyogenes. For example, the following Cas9 orthologs can be used inconnection with the prime editor constructs described in thisspecification. In addition, any variant Cas9 orthologs having at least80%, at least 85%, at least 90%, at least 95%, or at least 99% sequenceidentity to any of the below orthologs may also be used with the presentprime editors.

Description Sequence LfCas9MKEYHIGLDIGTSSIGWAVTDSQFKLMRIKGKTAIGVRLFEEGKTAAERR LactobacillusTFRTTRRRLKRRKWRLHYLDEIFAPHLQEVDENFLRRLKQSNIHPEDPTK fermentumNQAFIGKLLFPDLLKKNERGYPTLIKMRDELPVEQRAHYPVMNIYKLRE wild typeAMINEDRQFDLREVYLAVHHIVKYRGHFLNNASVDKFKVGRIDFDKSFN GenBank:VLNEAYEELQNGEGSFTIEPSKVEKIGQLLLDTKMRKLDRQKAVAKLLE SNX31424.11VKVADKEETKRNKQIATAMSKLVLGYKADFATVAMANGNEWKIDLSSETSEDEIEKFREELSDAQNDILTEITSLFSQIMLNEIVPNGMSISESMMDRYWTHERQLAEVKEYLATQPASARKEFDQVYNKYIGQAPKERGFDLEKGLKKILSKKENWKEIDELLKAGDFLPKQRTSANGVIPHQMHQQELDRIIEKQAKYYPWLATENPATGERDRHQAKYELDQLVSFRIPYYVGPLVTPEVQKATSGAKFAWAKRKEDGEITPWNLWDKIDRAESAEAFIKRMTVKDTYLLNEDVLPANSLLYQKYNVLNELNNVRVNGRRLSVGIKQDIYTELFKKKKTVKASDVASLVMAKTRGVNKPSVEGLSDPKKFNSNLATYLDLKSIVGDKVDDNRYQTDLENIIEWRSVFEDGEIFADKLTEVEWLTDEQRSALVKKRYKGWGRLSKKLLTGIVDENGQRIIDLMWNTDQNFKEIVDQPVFKEQIDQLNQKAITNDGMTLRERVESVLDDAYTSPQNKKAIWQVVRVVEDIVKAVGNAPKSISIEFARNEGNKGEITRSRRTQLQKLFEDQAHELVKDTSLTEELEKAPDLSDRYYFYFTQGGKDMYTGDPINFDEISTKYDIDHILPQSFVKDNSLDNRVLTSRKENNKKSDQVPAKLYAAKMKPYWNQLLKQGLITQRKFENLTKDVDQNIKYRSLGFVKRQLVETRQVIKLTANILGSMYQEAGTEIIETRAGLTKQLREEFDLPKVREVNDYHHAVDAYLTTFAGQYLNRRYPKLRSFFVYGEYMKFKHGSDLKLRNFNFFHELMEGDKSQGKVVDQQTGELITTRDEVAKSFDRLLNMKYMLVSKEVHDRSDQLYGATIVTAKESGKLTSPIEIKKNRLVDLYGAYTNGTSAFMTIIKFTGNKPKYKVIGIPTTSAASLKRAGKPGSESYNQELHRIIKSNPKVKKGFEIVVPHVSYGQLIVDGDCKFTLASPTVQHPATQLVLSKKSLETISSGYKILKDKPAIANERLIRVFDEVVGQMNRYFTIFDQRSNRQKVADARDKFLSLPTESKYEGAKKVQVGKTEVITNLLMGLHANATQGDLKVLGLATFGFFQSTTGLSLSEDTMIVYQSPTGLFERRICLK DI (SEQ ID NO: 26)SaCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG StaphylococcusALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH aureusRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA wild typeDLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN GenBank:PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP AYD60528.1NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 27) SaCas9MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRS StaphylococcusKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQ aureusKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKK (SEQ ID NO: 28) StCas9MLFNKCIIISINLDFSNKEKCMTKPYSIGLDIGTNSVGWAVITDNYKVPSK StreptococcusKMKVLGNTSKKYIKKNLLGVLLFDSGITAEGRRLKRTARRRYTRRRNRI thermophilusLYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEEKVYH UniProtKB/DEFPTIYHLRKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNSKN Swiss-Prot:NDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIVKDKISKLEKKDRILKLF G3ECR1.2PGEKNSGIFSEFLKLIVGNQADFRKCFNLDEKASLHFSKESYDEDLETLL Wild typeGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAPLSSAMIKRYNEHKEDLALLKEYIRNISLKTYNEVFKDDTKNGYAGYIDGKTNQEDFYVYLKNLLAEFEGADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQEMRAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLARGNSDFAWSIRKRNEKITPWNFEDVIDKESSAEAFINRMTSFDLYLPEEKVLPKHSLLYETFNVYNELTKVRFIAESMRDYQFLDSKQKKDIVRLYFKDKRKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLSTYHDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLIDDGISNRNFMQLIHDDALSFKKKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRKPESIVVEMARENQYTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNGKDMYTGDDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDFPSLEVVKKRKTFWYQLLKSKLISQRKFDNLTKAERGGLLPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKDENNRAVRTVKIITLKSTLVSQFRKDFELYKVREINDFHHAHDAYLNAVIASALLKKYPKLEPEFVYGDYPKYNSFRERKSATEKVYFYSNIMNIFKKSISLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLSYPQVNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKPNSNENLVGAKEYLDPKKYGGYAGISNSFAVLVKGTIEKGAKKKITNVLEFQGISILDRINYRKDKLNFLLEKGYKDIELIIELPKYSLFELSDGSRRMLASILSTNNKRGEIHKGNQIFLSQKFVKLLYHAKRISNTINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNSAFQSWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFEFLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRIDLAKLGEG (SEQ ID NO: 29) LcCas9MKIKNYNLALTPSTSAVGHVEVDDDLNILEPVHHQKAIGVAKFGEGETA LactobacillusEARRLARSARRTTKRRANRINHYFNEIMKPEIDKVDPLMFDRIKQAGLSP crispatusLDERKEFRTVIFDRPNIASYYHNQFPTIWHLQKYLMITDEKADIRLIYWA NCBILHSLLKHRGHFFNTTPMSQFKPGKLNLKDDMLALDDYNDLEGLSFAVA ReferenceNSPEIEKVIKDRSMHKKEKIAELKKLIVNDVPDKDLAKRNNKIITQIVNAI Sequence:MGNSFHLNFIFDMDLDKLTSKAWSFKLDDPELDTKFDAISGSMTDNQIGI WP_133478044.1FETLQKIYSAISLLDILNGSSNVVDAKNALYDKHKRDLNLYFKFLNTLPD Wild typeEIAKTLKAGYTLYIGNRKKDLLAARKLLKVNVAKNFSQDDFYKLINKELKSIDKQGLQTRFSEKVGELVAQNNFLPVQRSSDNVFIPYQLNAITFNKILENQGKYYDFLVKPNPAKKDRKNAPYELSQLMQFTIPYYVGPLVTPEEQVKSGIPKTSRFAWMVRKDNGAITPWNFYDKVDIEATADKFIKRSIAKDSYLLSELVLPKHSLLYEKYEVFNELSNVSLDGKKLSGGVKQILFNEVFKKTNKVNTSRILKALAKHNIPGSKITGLSNPEEFTSSLQTYNAWKKYFPNQIDNFAYQQDLEKMIEWSTVFEDHKILAKKLDEIEWLDDDQKKFVANTRLRGWGRLSKRLLTGLKDNYGKSIMQRLETTKANFQQIVYKPEFREQIDKISQAAAKNQSLEDILANSYTSPSNRKAIRKTMSVVDEYIKLNHGKEPDKIFLMFQRSEQEKGKQTEARSKQLNRILSQLKADKSANKLFSKQLADEFSNAIKKSKYKLNDKQYFYFQQLGRDALTGEVIDYDELYKYTVLHIIPRSKLTDDSQNNKVLTKYKIVDGSVALKFGNSYSDALGMPIKAFWTELNRLKLIPKGKLLNLTTDFSTLNKYQRDGYIARQLVETQQIVKLLATIMQSRFKHTKIIEVRNSQVANIRYQFDYFRIKNLNEYYRGFDAYLAAVVGTYLYKVYPKARRLFVYGQYLKPKKTNQENQDMHLDSEKKSQGFNFLWNLLYGKQDQIFVNGTDVIAFNRKDLITKMNTVYNYKSQKISLAIDYHNGAMFKATLFPRNDRDTAKTRKLIPKKKDYDTDIYGGYTSNVDGYMLLAEIIKRDGNKQYGFYGVPSRLVSELDTLKKTRYTEYEEKLKEIIKPELGVDLKKIKKIKILKNKVPFNQVIIDKGSKFFITSTSYRWNYRQLILSAESQQTLMDLVVDPDFSNHKARKDARKNADERLIKVYEEILYQVKNYMPMFVELHRCYEKLVDAQKTFKSLKISDKAMVLNQILILLHSNATSPVLEKLGYHTRFTLGKKHNLISENAVLVTQSITGLKENHVSIKQML (SEQ ID NO: 30) PdCas9MTNEKYSIGLDIGTSSIGFAVVNDNNRVIRVKGKNAIGVRLFDEGKAAA PedicoccusDRRSFRTTRRSFRTTRRRLSRRRWRLKLLREIFDAYITPVDEAFFIRLKES damnnosusNLSPKDSKKQYSGDILFNDRSDKDFYEKYPTIYHLRNALMTEHRKFDVR NCBIEIYLAIHHIMKFRGHFLNATPANNFKVGRLNLEEKFEELNDIYQRVFPDE ReferenceSIEFRTDNLEQIKEVLLDNKRSRADRQRTLVSDIYQSSEDKDIEKRNKAV Sequence:ATEILKASLGNKAKLNVITNVEVDKEAAKEWSITFDSESIDDDLAKIEGQ WP_062913273.1MTDDGHEIIEVLRSLYSGITLSAIVPENHTLSQSMVAKYDLHKDHLKLFK Wild typeKLINGMTDTKKAKNLRAAYDGYIDGVKGKVLPQEDFYKQVQVNLDDSAEANEIQTYIDQDIFMPKQRTKANGSIPHQLQQQELDQIIENQKAYYPWLAELNPNPDKKRQQLAKYKLDELVTFRVPYYVGPMITAKDQKNQSGAEFAWMIRKEPGNITPWNFDQKVDRMATANQFIKRMTTTDTYLLGEDVLPAQSLLYQKFEVLNELNKIRIDHKPISIEQKQQIFNDLFKQFKNVTIKHLQDYLVSQGQYSKRPLIEGLADEKRFNSSLSTYSDLCGIFGAKLVEENDRQEDLEKIIEWSTIFEDKKIYRAKLNDLTWLTDDQKEKLATKRYQGWGRLSRKLLVGLKNSEHRNIMDILWITNENFMQIQAEPDFAKLVTDANKGMLEKTDSQDVINDLYTSPQNKKAIRQILLVVHDIQNAMHGQAPAKIHVEFARGEERNPRRSVQRQRQVEAAYEKVSNELVSAKVRQEFKEAINNKRDFKDRLFLYFMQGGIDIYTGKQLNIDQLSSYQIDHILPQAFVKDDSLTNRVLTNENQVKADSVPIDIFGKKMLSVWGRMKDQGLISKGKYRNLTMNPENISAHTENGFINRQLVETRQVIKLAVNILADEYGDSTQIISVKADLSHQMREDFELLKNRDVNDYHHAFDAYLAAFIGNYLLKRYPKLESYFVYGDFKKFTQKETKMRRFNFIYDLKHCDQVVNKETGEILWTKDEDIKYIRHLFAYKKILVSHEVREKRGALYNQTIYKAKDDKGSGQESKKLIRIKDDKETKIYGGYSGKSLAYMTIVQITKKNKVSYRVIGIPTLALARLNKLENDSTENNGELYKIIKPQFTHYKVDKKNGEIIETTDDFKIVVSKVRFQQLIDDAGQFFMLASDTYKNNAQQLVISNNALKAINNTNITDCPRDDLERLDNLRLDSAFDEIVKKMDKYFSAYDANNFREKIRNSNLIFYQLPVEDQWENNKITELGKRTVLTRILQGLHANATTTDMSIFKIKTPFGQLRQRSGISLSENAQLIYQSPTGLFERRVQLNKIK (SEQ ID NO: 31)FnCas9 MKKQKFSDYYLGFDIGTNSVGWCVTDLDYNVLRFNKKDMWGSRLFEE FusobateriumAKTAAERRVQRNSRRRLKRRKWRLNLLEEIFSNEILKIDSNFFRRLKESSL nucleatumWLEDKSSKEKFTLFNDDNYKDYDFYKQYPTIFHLRNELIKNPEKKDIRLV NCBIYLAIHSIFKSRGHFLFEGQNLKEIKNFETLYNNLIAFLEDNGINKIIDKNNI ReferenceEKLEKIVCDSKKGLKDKEKEFKEIFNSDKQLVAIFKLSVGSSVSLNDLFD Sequence:TDEYKKGEVEKEKISFREQIYEDDKPIYYSILGEKIELLDIAKTFYDFMVL WP_060798984.1NNILADSQYISEAKVKLYEEHKKDLKNLKYIIRKYNKGNYDKLFKDKNENNYSAYIGLNKEKSKKEVIEKSRLKIDDLIKNIKGYLPKVEEIEEKDKAIFNKILNKIELKTILPKQRISDNGTLPYQIHEAELEKILENQSKYYDFLNYEENGIITKDKLLMTFKFRIPYYVGPLNSYHKDKGGNSWIVRKEEGKILPWNFEQKVDIEKSAEEFIKRMTNKCTYLNGEDVIPKDTFLYSEYVILNELNKVQVNDEFLNEENKRKIIDELFKENKKVSEKKFKEYLLVKQIVDGTIELKGVKDSFNSNYISYIRFKDIFGEKLNLDIYKEISEKSILWKCLYGDDKKIFEKKIKNEYGDILTKDEIKKINTFKFNNWGRLSEKLLTGIEFINLETGECYSSVMDALRRTNYNLMELLSSKFTLQESINNENKEMNEASYRDLIEESYVSPSLKRAIFQTLKIYEEIRKITGRVPKKVFIEMARGGDESMKNKKIPARQEQLKKLYDSCGNDIANFSIDIKEMKNSLISYDNNSLRQKKLYLYYLQFGKCMYTGREIDLDRLLQNNDTYDIDHIYPRSKVIKDDSFDNLVLVLKNENAEKSNEYPVKKEIQEKMKSFWRFLKEKNFISDEKYKRLTGKDDFELRGFMARQLVNVRQTTKEVGKILQQIEPEIKIVYSKAEIASSFREMFDFIKVRELNDTHHAKDAYLNIVAGNVYNTKFTEKPYRYLQEIKENYDVKKIYNYDIKNAWDKENSLEIVKKNMEKNTVNITRFIKEKKGQLFDLNPIKKGETSNEIISIKPKVYNGKDDKLNEKYGYYKSLNPAYFLYVEHKEKNKRIKSFERVNLVDVNNIKDEKSLVKYLIENKKLVEPRVIKKVYKRQVILINDYPYSIVTLDSNKLMDFENLKPLFLENKYEKILKNVIKFLEDNQGKSEENYKFIYLKKKDRYEKNETLESVKDRYNLEFNEMYDKFLEKLDSKDYKNYMNNKKYQELLDVKEKFIKLNLFDKAFTLKSFLDLFNRKTMADFSKVGLTKYLGKIQKISSNVLSKNELYLLEESVTGLFVKKIKL (SEQ ID NO: 32) EcCas9RRKQRIQILQELLGEEVLKTDPGFFHRMKESRYVVEDKRTLDGKQVELP EnterococcusYALFVDKDYTDKEYYKQFPTINHLIVYLMTTSDTPDIRLVYLALHYYMK cecorumNRGNFLHSGDINNVKDINDILEQLDNVLETFLDGWNLKLKSYVEDIKNIY NCBINRDLGRGERKKAFVNTLGAKTKAEKAFCSLISGGSTNLAELFDDSSLKEI ReferenceETPKIEFASSSLEDKIDGIQEALEDRFAVIEAAKRLYDWKTLTDILGDSSS Sequence:LAEARVNSYQMHHEQLLELKSLVKEYLDRKVFQEVFVSLNVANNYPAY WP_047338501.1IGHTKINGKKKELEVKRTKRNDFYSYVKKQVIEPIKKKVSDEAVLTKLSE Wild typeIESLIEVDKYLPLQVNSDNGVIPYQVKLNELTRIFDNLENRIPVLRENRDKIIKTFKFRIPYYVGSLNGVVKNGKCTNWMVRKEEGKIYPWNFEDKVDLEASAEQFIRRMTNKCTYLVNEDVLPKYSLLYSKYLVLSELNNLRIDGRPLDVKIKQDIYENVFKKNRKVTLKKIKKYLLKEGIITDDDELSGLADDVKSSLTAYRDFKEKLGHLDLSEAQMENIILNITLFGDDKKLLKKRLAALYPFIDDKSLNRIATLNYRDWGRLSERFLSGITSVDQETGELRTIIQCMYETQANLMQLLAEPYHFVEAIEKENPKVDLESISYRIVNDLYVSPAVKRQIWQTLLVIKDIKQVMKHDPERIFIEMAREKQESKKTKSRKQVLSEVYKKAKEYEHLFEKLNSLTEEQLRSKKIYLYFTQLGKCMYSGEPIDFENLVSANSNYDIDHIYPQSKTIDDSFNNIVLVKKSLNAYKSNHYPIDKNIRDNEKVKTLWNTLVSKGLITKEKYERLIRSTPFSDEELAGFIARQLVETRQSTKAVAEILSNWFPESEIVYSKAKNVSNFRQDFEILKVRELNDCHHAHDAYLNIVVGNAYHTKFTNSPYRFIKNKANQEYNLRKLLQKVNKIESNGVVAWVGQSENNPGTIATVKKVIRRNTVLISRMVKEVDGQLFDLTLMKKGKGQVPIKSSDERLTDISKYGGYNKATGAYFTFVKSKKRGKVVRSFEYVPLHLSKQFENNNELLKEYIEKDRGLTDVEILIPKVLINSLFRYNGSLVRITGRGDTRLLLVHEQPLYVSNSFVQQLKSVSSYKLKKSENDNAKLTKTATEKLSNIDELYDGLLRKLDLPIYSYWFSSIKEYLVESRTKYIKLSIEEKALVIFEILHLFQSDAQVPNLKILGLSTKPSRIRIQKNLKDTDKMSIIHQSPSGIFEHEIELTSL (SEQ ID NO: 33) AhCas9MQNGFLGITVSSEQVGWAVTNPKYELERASRKDLWGVRLFDKAETAED AnaerostipesRRMFRTNRRLNQRKKNRIHYLRDIFHEEVNQKDPNFFQQLDESNFCEDD hadrusRTVEFNFDTNLYKNQFPTVYHLRKYLMETKDKPDIRLVYLAFSKFMKN NCBIRGHFLYKGNLGEVMDFENSMKGFCESLEKFNIDFPTLSDEQVKEVRDIL ReferenceCDHKIAKTVKKKNIITITKVKSKTAKAWIGLFCGCSVPVKVLFQDIDEEIV Sequence:TDPEKISFEDASYDDYIANIEKGVGIYYEAIVSAKMLFDWSILNEILGDHQ WP_044924278.1LLSDAMIAEYNKHHDDLKRLQKIIKGTGSRELYQDIFINDVSGNYVCYV Wild typeGHAKTMSSADQKQFYTFLKNRLKNVNGISSEDAEWIDTEIKNGTLLPKQTKRDNSVIPHQLQLREFELILDNMQEMYPFLKENREKLLKIFNFVIPYYVGPLKGVVRKGESTNWMVPKKDGVIHPWNFDEMVDKEASAECFISRMTGNCSYLFNEKVLPKNSLLYETFEVLNELNPLKINGEPISVELKQRIYEQLFLTGKKVTKKSLTKYLIKNGYDKDIELSGIDNEFHSNLKSHIDFEDYDNLSDEEVEQIILRITVFEDKQLLKDYLNREFVKLSEDERKQICSLSYKGWGNLSEMLLNGITVTDSNGVEVSVMDMLWNTNLNLMQILSKKYGYKAEIEHYNKEHEKTIYNREDLMDYLNIPPAQRRKVNQLITIVKSLKKTYGVPNKIFFKISREHQDDPKRTSSRKEQLKYLYKSLKSEDEKHLMKELDELNDHELSNDKVYLYFLQKGRCIYSGKKLNLSRLRKSNYQNDIDYIYPLSAVNDRSMNNKVLTGIQENRADKYTYFPVDSEIQKKMKGFWMELVLQGFMTKEKYFRLSRENDFSKSELVSFIEREISDNQQSGRMIASVLQYYFPESKIVFVKEKLISSFKRDFHLISSYGHNHLQAAKDAYITIVVGNVYHTKFTMDPAIYFKNHKRKDYDLNRLFLENISRDGQIAWESGPYGSIQTVRKEYAQNHIAVTKRVVEVKGGLFKQMPLKKGHGEYPLKTNDPRFGNIAQYGGYTNVTGSYFVLVESMEKGKKRISLEYVPVYLHERLEDDPGHKLLKEYLVDHRKLNHPKILLAKVRKNSLLKIDGFYYRLNGRSGNALILTNAVELIMDDWQTKTANKISGYMKRRAIDKKARVYQNEFHIQELEQLYDFYLDKLKNGVYKNRKNNQAELIHNEKEQFMELKTEDQCVLLTEIKKLFVCSPMQADLTLIGGSKHTGMIAMSSNVTKADFAVIAEDPLGLRNKVIYSHKGEK (SEQ ID NO: 34) KvCas9MSQNNNKIYNIGLDIGDASVGWAVVDEHYNLLKRHGKHMWGSRLFTQ KandleriaANTAVERRSSRSTRRRYNKRRERIRLLREIMEDMVLDVDPTFFIRLANVS vitulinaFLDQEDKKDYLKENYHSNYNLFIDKDFNDKTYYDKYPTIYHLRKHLCES NCBIKEKEDPRLIYLALHHIVKYRGNFLYEGQKFSMDVSNIEDKMIDVLRQFN ReferenceEINLFEYVEDRKKIDEVLNVLKEPLSKKHKAEKAFALFDTTKDNKAAYK Sequence:ELCAALAGNKFNVTKMLKEAELHDEDEKDISFKFSDATFDDAFVEKQPL WP_031589969.1LGDCVEFIDLLHDIYSWVELQNILGSAHTSEPSISAAMIQRYEDHKNDLK Wild typeLLKDVIRKYLPKKYFEVFRDEKSKKNNYCNYINHPSKTPVDEFYKYIKKLIEKIDDPDVKTILNKIELESFMLKQNSRTNGAVPYQMQLDELNKILENQSVYYSDLKDNEDKIRSILTFRIPYYFGPLNITKDRQFDWIIKKEGKENERILPWNANEIVDVDKTADEFIKRMRNFCTYFPDEPVMAKNSLTVSKYEVLNEINKLRINDHLIKRDMKDKMLHTLFMDHKSISANAMKKWLVKNQYFSNTDDIKIEGFQKENACSTSLTPWIDFTKIFGKINESNYDFIEKIIYDVTVFEDKKILRRRLKKEYDLDEEKIKKILKLKYSGWSRLSKKLLSGIKTKYKDSTRTPETVLEVMERTNMNLMQVINDEKLGFKKTIDDANSTSVSGKFSYAEVQELAGSPAIKRGIWQALLIVDEIKKIMKHEPAHVYIEFARNEDEKERKDSFVNQMLKLYKDYDFEDETEKEANKHLKGEDAKSKIRSERLKLYYTQMGKCMYTGKSLDIDRLDTYQVDHIVPQSLLKDDSIDNKVLVLSSENQRKLDDLVIPSSIRNKMYGFWEKLFNNKIISPKKFYSLIKTEFNEKDQERFINRQIVETRQITKHVAQIIDNHYENTKVVTVRADLSHQFRERYHIYKNRDINDFHHAHDAYIATILGTYIGHRFESLDAKYIYGEYKRIFRNQKNKGKEMKKNNDGFILNSMRNIYADKDTGEIVWDPNYIDRIKKCFYYKDCFVTKKLEENNGTFFNVTVLPNDTNSDKDNTLATVPVNKYRSNVNKYGGFSGVNSFIVAIKGKKKKGKKVIEVNKLTGIPLMYKNADEEIKINYLKQAEDLEEVQIGKEILKNQLIEKDGGLYYIVAPTEIINAKQLILNESQTKLVCEIYKAMKYKNYDNLDSEKIIDLYRLLINKMELYYPEYRKQLVKKFEDRYEQLKVISIEEKCNIIKQILATLHCNSSIGKIMYSDFKISTTIGRLNGRTISLDDISFIAESPTGMYSKKYKL (SEQ ID NO: 35) EfCas9MRLFEEGHTAEDRRLKRTARRRISRRRNRLRYLQAFFEEAMTDLDENFF EnterococcusARLQESFLVPEDKKWHRHPIFAKLEDEVAYHETYPTIYHLRKKLADSSE faecalisQADLRLIYLALAHIVKYRGHFLIEGKLSTENTSVKDQFQQFMVIYNQTFV NCBINGESRLVSAPLPESVLIEEELTEKASRTKKSEKVLQQFPQEKANGLFGQF ReferenceLKLMVGNKADFKKVFGLEEEAKITYASESYEEDLEGILAKVGDEYSDVF Sequence:LAAKNVYDAVELSTILADSDKKSHAKLSSSMIVRFTEHQEDLKKFKRFIR WP_016631044.1ENCPDEYDNLFKNEQKDGYAGYIAHAGKVSQLKFYQYVKKIIQDIAGAE Wild typeYFLEKIAQENFLRKQRTFDNGVIPHQIHLAELQAIIHRQAAYYPFLKENQEKIEQLVTFRIPYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLDQSATAFIERMTNFDTYLPSEKVLPKHSLLYEKFMVFNELTKISYTDDRGIKANFSGKEKEKIFDYLFKTRRKVKKKDIIQFYRNEYNTEIVTLSGLEEDQFNASFSTYQDLLKCGLTRAELDHPDNAEKLEDIIKILTIFEDRQRIRTQLSTFKGQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESGKTILDYLVKDDGVSKHYNRNFMQLINDSQLSFKNAIQKAQSSEHEETLSETVNELAGSPAIKKGIYQSLKIVDELVAIMGYAPKRIVVEMARENQTTSTGKRRSIQRLKIVEKAMAEIGSNLLKEQPTTNEQLRDTRLFLYYMQNGKDMYTGDELSLHRLSHYDIDHIIPQSFMKDDSLDNLVLVGSTENRGKSDDVPSKEVVKDMKAYWEKLYAAGLISQRKFQRLTKGEQGGLTLEDKAHFIQRQLVETRQITKNVAGILDQRYNAKSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYHHGQDAYLNCVVATTLLKVYPNLAPEFVYGEYPKFQTFKENKATAKAIIYTNLLRFFTEDEPRFTKDGEILWSNSYLKTIKKELNYHQMNIVKKVEVQKGGFSKESIKPKGPSNKLIPVKNGLDPQKYGGFDSPVVAYTVLFTHEKGKKPLIKQEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPKYTLYEFPEGRRRLLASAKEAQKGNQMVLPEHLLTLLYHAKQCLLPNQSESLAYVEQHQPEFQEILERVVDFAEVHTLAKSKVQQIVKLFEANQTADVKEIAASFIQLMQFNAMGAPSTFKFFQKDIERARYTSIKEIFDATIIYQSPTGLYETRRKVVD (SEQ ID NO: 36)Staphylococcus KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR aureusGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL Cas9SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG (SEQ ID NO: 37) GeobacillusMKYKIGLDIGITSIGWAVINLDIPRIEDLGVRIFDRAENPKTGESLALPRRL thermodenitrificansARSARRRLRRRKHRLERIRRLFVREGILTKEELNKLFEKKHEIDVWQLRV Cas9EALDRKLNNDELARILLHLAKRRGFRSNRKSERTNKENSTMLKHIEENQSILSSYRTVAEMVVKDPKFSLHKRNKEDNYTNTVARDDLEREIKLIFAKQREYGNIVCTEAFEHEYISIWASQRPFASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFTVWEHINKLRLVSPGGIRALTDDERRLIYKQAFHKNKITFHDVRTLLNLPDDTRFKGLLYDRNTTLKENEKVRFLELGAYHKIRKAIDSVYGKGAAKSFRPIDFDTFGYALTMFKDDTDIRSYLRNEYEQNGKRMENLADKVYDEELIEELLNLSFSKFGHLSLKALRNILPYMEQGEVYSTACERAGYTFTGPKKKQKTVLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARELSQSFDERRKMQKEQEGNRKKNETAIRQLVEYGLTLNPTGLDIVKFKLWSEQNGKCAYSLQPIEIERLLEPGYTEVDHVIPYSRSLDDSYTNKVLVLTKENREKGNRTPAEYLGLGSERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEENEFKNRNLNDTRYISRFLANFIREHLKFADSDDKQKVYTVNGRITAHLRSRWNFNKNREESNLHHAVDAAIVACTTPSDIARVTAFYQRREQNKELSKKTDPQFPQPWPHFADELQARLSKNPKESIKALNLGNYDNEKLESLQPVFVSRMPKRSITGAAHQETLRRYIGIDERSGKIQTVVKKKLSEIQLDKTGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGELGPIIRTIKIIDTTNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPIYTIDMMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIEFPREKTIKTAVGEEIKIKDLFAYYQTIDSSNGGLSLVSHDNNFSLRSIGSRTLKRFEKYQVDVLGNIYKVRGEKRVGVASSSHSKAGETIRPL (SEQ ID NO: 38) ScCas9MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLM S. canisGALLFDSGETAEATRLKRTARRRYTRRKNRIRYLQEIFANEMAKLDDSF 1375 AAFQRLEESFLVEEDKKNERHPIFGNLADEVAYHRNYPTIYHLRKKLADSPE 159.2 kDaKADLRLIYLALAHIIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGNIIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLGQIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASMVKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYAGYVGIGIKHRKRTTKLATQEEFYKFIKPILEKMDGAEELLAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREKIEKILTFRIPYYVGPLARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPKHSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDGFSNRNFMQLIHDDSLTFKEEIEKAQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKELESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEADKAGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIREVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESILSKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEKGKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIKKELIFKLPKYSLFELENGRRRMLASATELQKANELVLPQHLVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDFSEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKYTSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSITGLYETRTDLSQLGGD (SEQ ID NO: 39)

The prime editors described herein may include any of the above Cas9ortholog sequences, or any variants thereof having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% sequence identitythereto.

The napDNAbp may include any suitable homologs and/or orthologs ornaturally occurring enzymes, such as, Cas9. Cas9 homologs and/ororthologs have been described in various species, including, but notlimited to, S. pyogenes and S. thermophilus. Preferably, the Cas moietyis configured (e.g, mutagenized, recombinantly engineered, or otherwiseobtained from nature) as a nickase, i.e., capable of cleaving only asingle strand of the target doubpdditional suitable Cas9 nucleases andsequences will be apparent to those of skill in the art based on thisdisclosure, and such Cas9 nucleases and sequences include Cas9 sequencesfrom the organisms and loci disclosed in Chylinski, Rhun, andCharpentier, “The tracrRNA and Cas9 families of type II CRISPR-Casimmunity systems” (2013) RNA Biology 10:5, 726-737; the entire contentsof which are incorporated herein by reference. In some embodiments, aCas9 nuclease has an inactive (e.g., an inactivated) DNA cleavagedomain, that is, the Cas9 is a nickase. In some embodiments, the Cas9protein comprises an amino acid sequence that is at least 80% identicalto the amino acid sequence of a Cas9 protein as provided by any one ofthe variants of Table 3. In some embodiments, the Cas9 protein comprisesan amino acid sequence that is at least 85%, at least 90%, at least 92%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orat least 99.5% identical to the amino acid sequence of a Cas9 protein asprovided by any one of the Cas9 orthologs in the above tables.

C. Dead Cas9 Variant

In certain embodiments, the prime editors described herein may include adead Cas9, e.g., dead SpCas9, which has no nuclease activity due to oneor more mutations that inactive both nuclease domains of Cas9, namelythe RuvC domain (which cleaves the non-protospacer DNA strand) and HNHdomain (which cleaves the protospacer DNA strand). The nucleaseinactivation may be due to one or mutations that result in one or moresubstitutions and/or deletions in the amino acid sequence of the encodedprotein, or any variants thereof having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto.

As used herein, the term “dCas9” refers to a nuclease-inactive Cas9 ornuclease-dead Cas9, or a functional fragment thereof, and embraces anynaturally occurring dCas9 from any organism, any naturally-occurringdCas9 equivalent or functional fragment thereof, any dCas9 homolog,ortholog, or paralog from any organism, and any mutant or variant of adCas9, naturally-occurring or engineered. The term dCas9 is not meant tobe particularly limiting and may be referred to as a “dCas9 orequivalent.” Exemplary dCas9 proteins and method for making dCas9proteins are further described herein and/or are described in the artand are incorporated herein by reference.

In other embodiments, dCas9 corresponds to, or comprises in part or inwhole, a Cas9 amino acid sequence having one or more mutations thatinactivate the Cas9 nuclease activity. In other embodiments, Cas9variants having mutations other than D10A and H840A are provided whichmay result in the full or partial inactivate of the endogenous Cas9nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations,by way of example, include other amino acid substitutions at D10 andH820, or other substitutions within the nuclease domains of Cas9 (e.g.,substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain)with reference to a wild type sequence such as Cas9 from Streptococcuspyogenes (NCBI Reference Sequence: NC_017053.1). In some embodiments,variants or homologues of Cas9 (e.g., variants of Cas9 fromStreptococcus pyogenes (NCBI Reference Sequence: NC_017053.1 (SEQ ID NO:20))) are provided which are at least about 70% identical, at leastabout 80% identical, at least about 90% identical, at least about 95%identical, at least about 98% identical, at least about 99% identical,at least about 99.5% identical, or at least about 99.9% identical toNCBI Reference Sequence: NC_017053.1. In some embodiments, variants ofdCas9 (e.g., variants of NCBI Reference Sequence: NC_017053.1 (SEQ IDNO: 20)) are provided having amino acid sequences which are shorter, orlonger than NC_017053.1 (SEQ ID NO: 20) by about 5 amino acids, by about10 amino acids, by about 15 amino acids, by about 20 amino acids, byabout 25 amino acids, by about 30 amino acids, by about 40 amino acids,by about 50 amino acids, by about 75 amino acids, by about 100 aminoacids or more.

In one embodiment, the dead Cas9 may be based on the canonical SpCas9sequence of Q99ZW2 and may have the following sequence, which comprisesa D10X and an H810X, wherein X may be any amino acid, substitutions(underlined and bolded), or a variant be variant of SEQ ID NO: 40 havingat least 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity thereto.

In one embodiment, the dead Cas9 may be based on the canonical SpCas9sequence of Q99ZW2 and may have the following sequence, which comprisesa D10A and an H810A substitutions (underlined and bolded), or be avariant of SEQ ID NO: 41 having at least 80%, at least 85%, at least90%, at least 95%, or at least 99% sequence identity thereto.

Description Sequence SEQ ID NO: dead Cas9 or MDKKYSIGL XIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO: dCas9RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 40 StreptococcusCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF pyogenesGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Q99ZW2AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE Cas9 withNPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF D10 X  andGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN H810 XLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS Where “X” isASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN any aminoGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE acidDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVD XIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDdead Cas9 or MDKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO: dCas9RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 41 StreptococcusCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF pyogenesGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Q99ZW2AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE Cas9 withNPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF D10 A  andGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN H810 ALLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVD AIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGD

D. Cas9 Nickase Variant

In one embodiment, the prime editors described herein comprise a Cas9nickase. The term “Cas9 nickase” of “nCas9” refers to a variant of Cas9which is capable of introducing a single-strand break in a double strandDNA molecule target. In some embodiments, the Cas9 nickase comprisesonly a single functioning nuclease domain. The wild type Cas9 (e.g., thecanonical SpCas9) comprises two separate nuclease domains, namely, theRuvC domain (which cleaves the non-protospacer DNA strand) and HNHdomain (which cleaves the protospacer DNA strand). In one embodiment,the Cas9 nickase comprises a mutation in the RuvC domain whichinactivates the RuvC nuclease activity. For example, mutations inaspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E)762, have been reported as loss-of-function mutations of the RuvCnuclease domain and the creation of a functional Cas9 nickase (e.g.,Nishimasu et al., “Crystal structure of Cas9 in complex with guide RNAand target DNA,” Cell 156(5), 935-949, which is incorporated herein byreference). Thus, nickase mutations in the RuvC domain could includeD10X, H983X, D986X, or E762X, wherein X is any amino acid other than thewild type amino acid. In certain embodiments, the nickase could be D10A,of H983A, or D986A, or E762A, or a combination thereof.

In various embodiments, the Cas9 nickase can having a mutation in theRuvC nuclease domain and have one of the following amino acid sequences,or a variant thereof having an amino acid sequence that has at least80%, at least 85%, at least 90%, at least 95%, or at least 99% sequenceidentity thereto.

Description Sequence SEQ ID NO: Cas9 nickase MDKKYSIGL XIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO: StreptococcusRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 42 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE D10 X ,NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF wherein X isGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN any alternateLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS amino acidASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 43 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE E762X,NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF wherein X isGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN any alternateLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS amino acidASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVI XMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 44 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE H983X,NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF wherein X isGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN any alternateLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS amino acidASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH XAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 45 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE D986X,NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF wherein X isGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN any alternateLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS amino acidASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH AH XAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 46 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE D10 ANPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 47 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE E762ANPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVI AMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 48 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE H983ANPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH AAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 49 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas 9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE D986ANPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH AH AAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGD

In another embodiment, the Cas9 nickase comprises a mutation in the HNHdomain which inactivates the HNH nuclease activity. For example,mutations in histidine (H) 840 or asparagine (R) 863 have been reportedas loss-of-function mutations of the HNH nuclease domain and thecreation of a functional Cas9 nickase (e.g., Nishimasu et al., “Crystalstructure of Cas9 in complex with guide RNA and target DNA,” Cell156(5), 935-949, which is incorporated herein by reference). Thus,nickase mutations in the HNH domain could include H840X and R863X,wherein X is any amino acid other than the wild type amino acid. Incertain embodiments, the nickase could be H840A or R863A or acombination thereof.

In various embodiments, the Cas9 nickase can have a mutation in the HNHnuclease domain and have one of the following amino acid sequences, or avariant thereof having an amino acid sequence that has at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% sequence identitythereto.

Description Sequence SEQ ID NO: Cas9 nickaseMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO: StreptococcusRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 50 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE H840 X ,NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF wherein X isGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN any alternateLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS amino acidASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVD XIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 51 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas 9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE H840 ANPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVD AIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 52 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE R863X,NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF wherein X isGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN any alternateLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS amino acidASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN X GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD SEQ ID NO:Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI 53 pyogenesCYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Q99ZW2GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9 withAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE R863 ANPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN A GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGD

In some embodiments, the N-terminal methionine is removed from a Cas9nickase, or from any Cas9 variant, ortholog, or equivalent disclosed orcontemplated herein. For example, methionine-minus Cas9 nickases includethe following sequences, or a variant thereof having an amino acidsequence that has at least 80%, at least 85%, at least 90%, at least95%, or at least 99% sequence identity thereto.

Description Sequence Cas9 nickaseDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG (Met minus)ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF StreptococcusFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST pyogenesDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ Q99ZW2LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Cas9 withSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA H840 X ,AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ wherein X isQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL any alternateVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK amino acidIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVD XIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 54) Cas9 nickaseDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG (Met minus)ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF StreptococcusFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST pyogenesDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ Q99ZW2LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Cas9 withSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA H840 AAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVD AIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 55) Cas9 nickaseDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG (Met minus)ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF StreptococcusFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST pyogenesDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ Q99ZW2LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Cas9 withSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA R863X,AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ wherein X isQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL any alternateVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK amino acidIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN X GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSTTGLYETRIDLSQLGGD (SEQ ID NO: 56) Cas9 nickaseDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG (Met minus)ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF StreptococcusFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST pyogenesDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ Q99ZW2LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Cas9 withSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA R863 AAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN A GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 57)

E. Other Cas9 Variants

Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins usedherein may also include other “Cas9 variants” having at least about 70%identical, at least about 80% identical, at least about 90% identical,at least about 95% identical, at least about 96% identical, at leastabout 97% identical, at least about 98% identical, at least about 99%identical, at least about 99.5% identical, or at least about 99.9%identical to any reference Cas9 protein, including any wild type Cas9,or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, orcircular permutant Cas9, or other variant of Cas9 disclosed herein orknown in the art. In some embodiments, a Cas9 variant may have 1, 2, 3,4, 5, 6, 7, 8,9, 10,11,12,13,14,15,16,17,18, 19, 20, 21, 22, 21, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to areference Cas9. In some embodiments, the Cas9 variant comprises afragment of a reference Cas9 (e.g., a gRNA binding domain or aDNA-cleavage domain), such that the fragment is at least about 70%identical, at least about 80% identical, at least about 90% identical,at least about 95% identical, at least about 96% identical, at leastabout 97% identical, at least about 98% identical, at least about 99%identical, at least about 99.5% identical, or at least about 99.9%identical to the corresponding fragment of wild type Cas9. In someembodiments, the fragment is at least 30%, at least 35%, at least 40%,at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% identical, at least 96%, at least 97%, at least 98%, at least99%, or at least 99.5% of the amino acid length of a corresponding wildtype Cas9 (e.g., SEQ ID NO: 18).

In some embodiments, the disclosure also may utilize Cas9 fragmentswhich retain their functionality and which are fragments of any hereindisclosed Cas9 protein. In some embodiments, the Cas9 fragment is atleast 100 amino acids in length. In some embodiments, the fragment is atleast 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least1300 amino acids in length.

In various embodiments, the prime editors disclosed herein may compriseone of the Cas9 variants described as follows, or a Cas9 variant thereofhaving at least about 70% identical, at least about 80% identical, atleast about 90% identical, at least about 95% identical, at least about96% identical, at least about 97% identical, at least about 98%identical, at least about 99% identical, at least about 99.5% identical,or at least about 99.9% identical to any reference Cas9 variants.

F. Small-Sized Cas9 Variants

In some embodiments, the prime editors contemplated herein can include aCas9 protein that is of smaller molecular weight than the canonicalSpCas9 sequence. In some embodiments, the smaller-sized Cas9 variantsmay facilitate delivery to cells, e.g., by an expression vector,nanoparticle, or other means of delivery. In certain embodiments, thesmaller-sized Cas9 variants can include enzymes categorized as type IIenzymes of the Class 2 CRISPR-Cas systems. In some embodiments, thesmaller-sized Cas9 variants can include enzymes categorized as type Venzymes of the Class 2 CRISPR-Cas systems. In other embodiments, thesmaller-sized Cas9 variants can include enzymes categorized as type VIenzymes of the Class 2 CRISPR-Cas systems.

The canonical SpCas9 protein is 1368 amino acids in length and has apredicted molecular weight of 158 kilodaltons. The term “small-sizedCas9 variant”, as used herein, refers to any Cas9 variant—naturallyoccurring, engineered, or otherwise—that is less than at least 1300amino acids, or at least less than 1290 amino acids, or than less than1280 amino acids, or less than 1270 amino acid, or less than 1260 aminoacid, or less than 1250 amino acids, or less than 1240 amino acids, orless than 1230 amino acids, or less than 1220 amino acids, or less than1210 amino acids, or less than 1200 amino acids, or less than 1190 aminoacids, or less than 1180 amino acids, or less than 1170 amino acids, orless than 1160 amino acids, or less than 1150 amino acids, or less than1140 amino acids, or less than 1130 amino acids, or less than 1120 aminoacids, or less than 1110 amino acids, or less than 1100 amino acids, orless than 1050 amino acids, or less than 1000 amino acids, or less than950 amino acids, or less than 900 amino acids, or less than 850 aminoacids, or less than 800 amino acids, or less than 750 amino acids, orless than 700 amino acids, or less than 650 amino acids, or less than600 amino acids, or less than 550 amino acids, or less than 500 aminoacids, but at least larger than about 400 amino acids and retaining therequired functions of the Cas9 protein. The Cas9 variants can includethose categorized as type II, type V, or type VI enzymes of the Class 2CRISPR-Cas system.

In various embodiments, the prime editors disclosed herein may compriseone of the small-sized Cas9 variants described as follows, or a Cas9variant thereof having at least about 70% identical, at least about 80%identical, at least about 90% identical, at least about 95% identical,at least about 96% identical, at least about 97% identical, at leastabout 98% identical, at least about 99% identical, at least about 99.5%identical, or at least about 99.9% identical to any referencesmall-sized Cas9 protein.

Description Sequence SEQ ID NO: SaCas9MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEA SEQ ID NO: StaphylococcusNVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLL 58 aureusTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRG 1053 AAVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLE 123 kDaRLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSI KKYSTDILGNLYEVKSKKHPQIIKKNmeCas9 MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDL SEQ ID NO: N.GVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRL 59 meningitidisLRTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAAL 1083 AADRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGA 124.5 kDaLLKGVAGNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDAALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGV KTALSFQKYQIDELGKEIRPCRLKKRPPVRCjCas9 MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKT SEQ ID NO: C. jejuniGESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLN 60 984 AAYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFA 114.9 kDaRVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK GeoCas9MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENP SEQ ID NO: G.QTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILT 61 stearothermophilusKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLL 1087 AAHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRT 127 kDaVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAVIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVG LASSAHSKPGKTIRPLQSTRD LbaCas12aMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVED SEQ ID NO: L. bacteriumEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYIS 62 1228 AALFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLF 143.9 kDaKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS VKH BhCas12bMATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILK SEQ ID NO: B. hisashiiLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQK 63 1108 AACNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSN 130.4 kDaKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSIS TIEDDSSKQSM

G. Cas9 Equivalents

In some embodiments, the prime editors described herein can include anyCas9 equivalent. As used herein, the term “Cas9 equivalent” is a broadterm that encompasses any napDNAbp protein that serves the same functionas Cas9 in the present prime editors despite that its amino acid primarysequence and/or its three-dimensional structure may be different and/orunrelated from an evolutionary standpoint. Thus, while Cas9 equivalentsinclude any Cas9 ortholog, homolog, mutant, or variant described orembraced herein that are evolutionarily related, the Cas9 equivalentsalso embrace proteins that may have evolved through convergent evolutionprocesses to have the same or similar function as Cas9, but which do notnecessarily have any similarity with regard to amino acid sequenceand/or three dimensional structure. The prime editors described hereembrace any Cas9 equivalent that would provide the same or similarfunction as Cas9 despite that the Cas9 equivalent may be based on aprotein that arose through convergent evolution. For instance, if Cas9refers to a type II enzyme of the CRISPR-Cas system, a Cas9 equivalentcan refer to a type V or type VI enzyme of the CRISPR-Cas system.

For example, Cas12e (CasX) is a Cas9 equivalent that reportedly has thesame function as Cas9 but which evolved through convergent evolution.Thus, the Cas12e (CasX) protein described in Liu et al., “CasX enzymescomprises a distinct family of RNA-guided genome editors,” Nature, 2019,Vol. 566: 218-223, is contemplated to be used with the prime editorsdescribed herein. In addition, any variant or modification of Cas12e(CasX) is conceivable and within the scope of the present disclosure.

Cas9 is a bacterial enzyme that evolved in a wide variety of species.However, the Cas9 equivalents contemplated herein may also be obtainedfrom archaea, which constitute a domain and kingdom of single-celledprokaryotic microbes different from bacteria.

In some embodiments, Cas9 equivalents may refer to Cas12e (CasX) orCas12d (CasY), which have been described in, for example, Burstein etal., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is herebyincorporated by reference. Using genome-resolved metagenomics, a numberof CRISPR-Cas systems were identified, including the first reported Cas9in the archaeal domain of life. This divergent Cas9 protein was found inlittle-studied nanoarchaea as part of an active CRISPR-Cas system. Inbacteria, two previously unknown systems were discovered, CRISPR-Cas12eand CRISPR-Cas12d, which are among the most compact systems yetdiscovered. In some embodiments, Cas9 refers to Cas12e, or a variant ofCas12e. In some embodiments, Cas9 refers to a Cas12d, or a variant ofCas12d. It should be appreciated that other RNA-guided DNA bindingproteins may be used as a nucleic acid programmable DNA binding protein(napDNAbp), and are within the scope of this disclosure. Also see Liu etal., “CasX enzymes comprises a distinct family of RNA-guided genomeeditors,” Nature, 2019, Vol. 566: 218-223. Any of these Cas9 equivalentsare contemplated.

In some embodiments, the Cas9 equivalent comprises an amino acidsequence that is at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% identical to anaturally-occurring Cas12e (CasX) or Cas12d (CasY) protein. In someembodiments, the napDNAbp is a naturally-occurring Cas12e (CasX) orCas12d (CasY) protein. In some embodiments, the napDNAbp comprises anamino acid sequence that is at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical to awild-type Cas moiety or any Cas moiety provided herein.

In various embodiments, the nucleic acid programmable DNA bindingproteins include, without limitation, Cas9 (e.g., dCas9 and nCas9),Cas12e (CasX), Cas12d (CasY), Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a(C2c2), Cas12c (C2c3), Argonaute, and Cas12b1. One example of a nucleicacid programmable DNA-binding protein that has different PAM specificitythan Cas9 is Clustered Regularly Interspaced Short Palindromic Repeatsfrom Prevotella and Francisella 1 (i.e, Cas12a (Cpf1)). Similar to Cas9,Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member oftype V subgroup of enzymes, rather than the type II subgroup. It hasbeen shown that Cas12a (Cpf1) mediates robust DNA interference withfeatures distinct from Cas9. Cas12a (Cpf1) is a single RNA-guidedendonuclease lacking tracrRNA, and it utilizes a T-richprotospacer-adjacent motif (TTN, TITN, or YTN). Moreover, Cpf1 cleavesDNA via a staggered DNA double-stranded break. Out of 16 Cpf1-familyproteins, two enzymes from Acidaminococcus and Lachnospiraceae are shownto have efficient genome-editing activity in human cells. Cpf1 proteinsare known in the art and have been described previously, for exampleYamano et al., “Crystal structure of Cpf1 in complex with guide RNA andtarget DNA.” Cell (165) 2016, p. 949-962; the entire contents of whichis hereby incorporated by reference.

In still other embodiments, the Cas protein may include any CRISPRassociated protein, including but not limited to, Cas12a, Cas12b1, Cas1,Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known asCsn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, andpreferably comprising a nickase mutation (e.g., a mutation correspondingto the D10A mutation of the wild type Cas9 polypeptide of SEQ ID NO:18).

In various other embodiments, the napDNAbp can be any of the followingproteins: a Cas9, a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), aCas12b1 (C2c1), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, aCas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, aCsn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or anArgonaute (Ago) domain, or a variant thereof.

Exemplary Cas9 equivalent protein sequences can include the following:

Description Sequence AsCas12aMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKEL previouslyKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQA known asTYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTV Cpf1)TTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPK AcidaminococcusFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQ sp.TQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIP (strainLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNE BV3L6)LNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKE UniProtKBKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL U2UMQ6KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 64) AsCas12aMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKEL nickaseKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQA (e.g.,TYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTV R1226A)TTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMANSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 65) LbCas12aMNYKTGLEDFIGKESLSKTLRNALIPTESTKIHMEEMGVIRDDELRAEKQ (previouslyQELKEIMDDYYRTFIEEKLGQIQGIQWNSLFQKMEETMEDISVRKDLDKI known asQNEKRKEICCYFTSDKRFKDLFNAKLITDILPNFIKDNKEYTEEEKAEKE Cpf1)QTRVLFQRFATAFTNYFNQRRNNFSEDNISTAISFRIVNENSEIHLQNMR LachnospiraceaeAFQRIEQQYPEEVCGMEEEYKDMLQEWQMKHIYSVDFYDRELTQPGIE bacteriumYYNGICGKINEHMNQFCQKNRINKNDFRMKKLHKQILCKKSSYYEIPFR GAM79FESDQEVYDALNEFIKTMKKKEIIRRCVHLGQECDDYDLGKIYISSNKYE Ref Seq.QISNALYGSWDTIRKCIKEEYMDALPGKGEKKEEKAEAAAKKEEYRSIA WP_119623382.1DIDKIISLYGSEMDRTISAKKCITEICDMAGQISIDPLVCNSDIKLLQNKEKTTEIKTILDSFLHVYQWGQTFIVSDIIEKDSYFYSELEDVLEDFEGITTLYNHVRSYVTQKPYSTVKFKLHFGSPTLANGWSQSKEYDNNAILLMRDQKFYLGIFNVRNKPDKQIIKGHEKEEKGDYKKMIYNLLPGPSKMLPKVFITSRSGQETYKPSKHILDGYNEKRHIKSSPKFDLGYCWDLIDYYKECIHKHPDWKNYDFHFSDTKDYEDISGFYREVEMQGYQIKWTYISADEIQKLDEKGQIFLFQIYNKDFSVHSTGKDNLHTMYLKNLFSEENLKDIVLKLNGEAELFFRKASIKTPIVHKKGSVLVNRSYTQTVGNKEIRVSIPEEYYTEIYNYLNHIGKGKLSSEAQRYLDEGKIKSFTATKDIVKNYRYCCDHYFLHLPITINFKAKSDVAVNERTLAYIAKKEDIHIIGIDRGERNLLYISVVDVHGNIREQRSFNIVNGYDYQQKLKDREKSRDAARKNWEEIEKIKELKEGYLSMVIHYIAQLVVKYNAVVAMEDLNYGFKTGRFKVERQVYQKFETMLIEKLHYLVFKDREVCEEGGVLRGYQLTYIPESLKKVGKQCGFIFYVPAGYTSKIDPTTGFVNLFSFKNLTNRESRQDFVGKFDEIRYDRDKKMFEFSFDYNNYIKKGTILASTKWKVYTNGTRLKRIVVNGKYTSQSMEVELTDAMEKMLQRAGIEYHDGKDLKGQIVEKGIEAEIIDIFRLTVQMRNSRSESEDREYDRLISPVLNDKEFFDTATADKTLPQDADANGAYCIALKGLYEVKQIKENWKENEQFPRNKLVQDNKTWFDFMQKKRYL (SEQ ID NO: 66) PcCas12a -MAKNFEDFKRLYSLSKTLRFEAKPIGATLDNIVKSGLLDEDEHRAASYV previouslyKVKKLIDEYHKVFIDRVLDDGCLPLENKGNNNSLAEYYESYVSRAQDE known atDAKKKFKEIQQNLRSVIAKKLTEDKAYANLFGNKLIESYKDKEDKKKII Cpf1DSDLIQFINTAESTQLDSMSQDEAKELVKEFWGFVTYFYGFFDNRKNMY PrevotellaTAEEKSTGIAYRLVNENLPKFIDNIEAFNRAITRPEIQENMGVLYSDFSEY copriLNVESIQEMFQLDYYNMLLTQKQIDVYNAIIGGKTDDEHDVKIKGINEYI Ref Seq.NLYNQQHKDDKLPKLKALFKQILSDRNAISWLPEEFNSDQEVLNAIKDC WP_119227726.1YERLAENVLGDKVLKSLLGSLADYSLDGIFIRNDLQLTDISQKMFGNWGVIQNAIMQNIKRVAPARKHKESEEDYEKRIAGIFKKADSFSISYINDCLNEADPNNAYFVENYFATFGAVNTPTMQRENLFALVQNAYTEVAALLHSDYPTVKHLAQDKANVSKIKALLDAIKSLQHFVKPLLGKGDESDKDERFYGELASLWAELDTVTPLYNMIRNYMTRKPYSQKKIKLNFENPQLLGGWDANKEKDYATIILRRNGLYYLAIMDKDSRKLLGKAMPSDGECYEKMVYKFFKDVTTMIPKCSTQLKDVQAYFKVNTDDYVLNSKAFNKPLTITKEVFDLNNVLYGKYKKFQKGYLTATGDNVGYTHAVNVWIKFCMDFLNSYDSTCIYDFSSLKPESYLSLDAFYQDANLLLYKLSFARASVSYINQLVEEGKMYLFQIYNKDFSEYSKGTPNMHTLYWKALFDERNLADVVYKLNGQAEMFYRKKSIENTHPTHPANHPILNKNKDNKKKESLFDYDLIKDRRYTVDKFMFHVPITMNFKSVGSENINQDVKAYLRHADDMHIIGIDRGERHLLYLVVIDLQGNIKEQYSLNEIVNEYNGNTYHTNYHDLLDVREEERLKARQSWQTIENIKELKEGYLSQVIHKITQLMVRYHAIVVLEDLSKGFMRSRQKVEKQVYQKFEKMLIDKLNYLVDKKTDVSTPGGLLNAYQLTCKSDSSQKLGKQSGFLFYIPAWNTSKIDPVTGFVNLLDTHSLNSKEKIKAFFSKFDAIRYNKDKKWFEFNLDYDKFGKKAEDTRTKWTLCTRGMRIDTFRNKEKNSQWDNQEVDLTTEMKSLLEHYYIDIHGNLKDAISAQTDKAFFTGLLHILKLTLQMRNSITGTETDYLVSPVADENGIFYDSRSCGNQLPENADANGAYNIARKGLMLIEQIKNAEDLNNVKFDISNKAWLNFAQQKPYKNG (SEQ ID NO: 67) ErCas12a -MFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRA previouslyNCFSANDISSSSCHRIVNDNAEIFFSNALVYRRIVKNLSNDDINKISGDMK known atDSLKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNLFMNLYCQKNKEN Cpf1KNLYKLRKLHKQILCIADTSYEVPYKFESDEEVYQSVNGFLDNISSKHIV EubacteriumERLRKIGENYNGYNLDKIYIVSKFYESVSQKTYRDWETINTALEIHYNNI rectaleLPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCPDDNIKAETYIH Ref Seq.EISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFM WP_119223642.1TEELVDKDNNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNNAIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSKTGVETYKPSAYILEGYKQNKHLKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDTSTYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSSGNDNLHTMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGNIQIVRKTIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYFLHMPITINFKANKTSFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKSFNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIAMEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSDKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTDINWRDGHDLRQDIIDYEIVQHIFEIFKLTVQMRNSLSELEDRDYDRLISPVLNENNIFYDSAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNKRYL (SEQ ID NO: 68) CsCas12a -MNYKTGLEDFIGKESLSKTLRNALIPTESTKIHMEEMGVIRDDELRAEKQ previouslyQELKEIMDDYYRAFIEEKLGQIQGIQWNSLFQKMEETMEDISVRKDLDKI known atQNEKRKEICCYFTSDKRFKDLFNAKLITDILPNFIKDNKEYTEEEKAEKE Cpf1QTRVLFQRFATAFTNYFNQRRNNFSEDNISTAISFRIVNENSEIHLQNMR ClostridiumAFQRIEQQYPEEVCGMEEEYKDMLQEWQMKHIYLVDFYDRVLTQPGIE sp. AF34-YYNGICGKINEHMNQFCQKNRINKNDFRMKKLHKQILCKKSSYYEIPFR 10BHFESDQEVYDALNEFIKTMKEKEIICRCVHLGQKCDDYDLGKIYISSNKYE Ref Seq.QISNALYGSWDTIRKCIKEEYMDALPGKGEKKEEKAEAAAKKEEYRSIA WP_118538418.1DIDKIISLYGSEMDRTISAKKCITEICDMAGQISTDPLVCNSDIKLLQNKEKTTEIKTILDSFLHVYQWGQTFIVSDIIEKDSYFYSELEDVLEDFEGITTLYNHVRSYVTQKPYSTVKFKLHFGSPTLANGWSQSKEYDNNAILLMRDQKFYLGIFNVRNKPDKQIIKGHEKEEKGDYKKMIYNLLPGPSKMLPKVFITSRSGQETYKPSKHILDGYNEKRHIKSSPKFDLGYCWDLIDYYKECIHKHPDWKNYDFHFSDTKDYEDISGFYREVEMQGYQIKWTYISADEIQKLDEKGQIFLFQIYNKDFSVHSTGKDNLHTMYLKNLFSEENLKDIVLKLNGEAELFFRKASIKTPVVHKKGSVLVNRSYTQTVGDKEIRVSIPEEYYTEIYNYLNHIGRGKLSTEAQRYLEERKIKSFTATKDIVKNYRYCCDHYFLHLPITINFKAKSDIAVNERTLAYIAKKEDIHIIGIDRGERNLLYISVVDVHGNIREQRSFNIVNGYDYQQKLKDREKSRDAARKNWEEIEKIKELKEGYLSMVIHYIAQLVVKYNAVVAMEDLNYGFKTGRFKVERQVYQKFETMLIEKLHYLVFKDREVCEEGGVLRGYQLTYIPESLKKVGKQCGFIFYVPAGYTSKIDPTTGFVNLFSFKNLTNRESRQDFVGKFDEIRYDRDKKMFEFSFDYNNYIKKGTMLASTKWKVYTNGTRLKRIVVNGKYTSQSMEVELTDAMEKMLQRAGIEYHDGKDLKGQIVEKGIEAEIIDIFRLTVQMRNSRSESEDREYDRLISPVLNDKGEFFDTATADKTLPQDADANGAYCIALKGLYEVKQIKENWKENEQFPRNKLVQDNKTWFDFMQKKRYL (SEQ ID NO: 69) BhCas12bMATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHH BacillusEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILREL hisashiiYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNL Ref Seq.KIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPI WP_095142515.1VKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSM (SEQ ID NO: 70) ThCas12bMSEKTTQRAYTLRLNRASGECAVCQNNSCDCWHDALWATHKAVNRG ThermomonasAKAFGDWLLTLRGGLCHTLVEMEVPAKGNNPPQRPTDQERRDRRVLLA hydrothermalisLSWLSVEDEHGAPKEFIVATGRDSADDRAKKVEEKLREILEKRDFQEHEI Ref Seq.DAWLQDCGPSLKAHIREDAVWVNRRALFDAAVERIKTLTWEEAWDFL WP_072754838EPFFGTQYFAGIGDGKDKDDAEGPARQGEKAKDLVQKAGQWLSARFGIGTGADFMSMAEAYEKIAKWASQAQNGDNGKATIEKLACALRPSEPPTLDTVLKCISGPGHKSATREYLKTLDKKSTVTQEDLNQLRKLADEDARNCRKKVGKKGKKPWADEVLKDVENSCELTYLQDNSPARHREFSVMLDHAARRVSMAHSWIKKAEQRRRQFESDAQKLKNLQERAPSAVEWLDRFCESRSMTTGANTGSGYRIRKRAIEGWSYVVQAWAEASCDTEDKRIAAARKVQADPEIEKFGDIQLFEALAADEAICVWRDQEGTQNPSILIDYVTGKTAEHNQKRFKVPAYRHPDELRHPVFCDFGNSRWSIQFAIHKEIRDRDKGAKQDTRQLQNRHGLKMRLWNGRSMTDVNLHWSSKRLTADLALDQNPNPNPTEVTRADRLGRAASSAFDHVKIKNVFNEKEWNGRLQAPRAELDRIAKLEEQGKTEQAEKLRKRLRWYVSFSPCLSPSGPFIVYAGQHNIQPKRSGQYAPHAQANKGRARLAQLILSRLPDLRILSVDLGHRFAAACAVWETLSSDAFRREIQGLNVLAGGSGEGDLFLHVEMTGDDGKRRTVVYRRIGPDQLLDNTPHPAPWARLDRQFLIKLQGEDEGVREASNEELWTVHKLEVEVGRTVPLIDRMVRSGFGKTEKQKERLKKLRELGWISAMPNEPSAETDEKEGEIRSISRSVDELMSSALGTLRLALKRHGNRARIAFAMTADYKPMPGGQKYYFHEAKEASKNDDETKRRDNQIEFLQDALSLWHDLFSSPDWEDNEAKKLWQNHIATLPNYQTPEEISAELKRVERNKKRKENRDKLRTAAKALAENDQLRQHLHDTWKERWESDDQQWKERLRSLKDWIFPRGKAEDNPSIRHVGGLSITRINTISGLYQILKAFKMRPEPDDLRKNIPQKGDDELENFNRRLLEARDRLREQRVKQLASRIIEAALGVGRIKIPKNGKLPKRPRTTVDTPCHAVVIESLKTYRPDDLRTRRENRQLMQWSSAKVRKYLKEGCELYGLHFLEVPANYTSRQCSRTGLPGIRCDDVPTGDFLKAPWWRRAINTAREKNGGDAKDRFLVDLYDHLNNLQSKGEALPATVRVPRQGGNLFIAGAQLDDTNKERRAIQADLNAAANIGLRALLDPDWRGRWWYVPCKDGTSEPALDRIEGSTAFNDVRSLPTGDNSSRRAPREIENLWRDPSGDSLESGTWSPTRAYWDTVQSRVIELLRRHAGLPTS (SEQ ID NO: 71) LsCas12bMSIRSFKLKLKTKSGVNAEQLRRGLWRTHQLINDGIAYYMNWLVLLRQ LaceyellaEDLFIRNKETNEIEKRSKEEIQAVLLERVHKQQQRNQWSGEVDEQTLLQ sacchariALRQLYEEIVPSVIGKSGNASLKARFFLGPLVDPNNKTTKDVSKSGPTPK WP_132221894.1WKKMKDAGDPNWVQEYEKYMAERQTLVRLEEMGLIPLFPMYTDEVGDIHWLPQASGYTRTWDRDMFQQAIERLLSWESWNRRVRERRAQFEKKTHDFASRFSESDVQWMNKLREYEAQQEKSLEENAFAPNEPYALTKKALRGWERVYHSWMRLDSAASEEAYWQEVATCQTAMRGEFGDPAIYQFLAQKENHDIWRGYPERVIDFAELNHLQRELRRAKEDATFTLPDSVDHPLWVRYEAPGGTNIHGYDLVQDTKRNLTLILDKFILPDENGSWHEVKKVPFSLAKSKQFHRQVWLQEEQKQKKREVVFYDYSTNLPHLGTLAGAKLQWDRNFLNKRTQQQIEETGEIGKVFFNISVDVRPAVEVKNGRLQNGLGKALTVLTHPDGTKIVTGWKAEQLEKWVGESGRVSSLGLDSLSEGLRVMSIDLGQRTSATVSVFEITKEAPDNPYKFFYQLEGTEMFAVHQRSFLLALPGENPPQKIKQMREIRWKERNRIKQQVDQLSAILRLHKKVNEDERIQAIDKLLQKVASWQLNEEIATAWNQALSQLYSKAKENDLQWNQAIKNAHHQLEPVVGKQISLWRKDLSTGRQGIAGLSLWSIEELEATKKLLTRWSKRSREPGVVKRIERFETFAKQIQHHINQVKENRLKQLANLIVMTALGYKYDQEQKKWIEVYPACQVVLFENLRSYRFSFERSRRENKKLMEWSHRSIPKLVQMQGELFGLQVADVYAAYSSRYHGRTGAPGIRCHALTEADLRNETNIIHELIEAGFIKEEHRPYLQQGDLVPWSGGELFATLQKPYDNPRILTLHADINAAQNIQKRFWHPSMWFRVNCESVMEGEIVTYVPKNKTVHKKQGKTFRFVKVEGSDVYEWAKWSKNRNKNTFSSITERKPPSSMILFRDPSGTFFKEQEWVEQKTFWGKVQSMIQAYMKKTIVQRMEE (SEQ ID NO: 72) DtCas12bMVLGRKDDTAELRRALWTTHEHVNLAVAEVERVLLRCRGRSYWTLDR DsulfonatronumRGDPVHVPESQVAEDALAMAREAQRRNGWPVVGEDEEILLALRYLYEQ thiodisrnutansIVPSCLLDDLGKPLKGDAQKIGTNYAGPLFDSDTCRRDEGKDVACCGPF WP_031386437HEVAGKYLGALPEWATPISKQEFDGKDASHLRFKATGGDDAFFRVSIEKANAWYEDPANQDALKNKAYNKDDWKKEKDKGISSWAVKYIQKQLQLGQDPRTEVRRKLWLELGLLPLFIPVFDKTMVGNLWNRLAVRLALAHLLSWESWNHRAVQDQALARAKRDELAALFLGMEDGFAGLREYELRRNESIKQHAFEPVDRPYVVSGRALRSWTRVREEWLRHGDTQESRKNICNRLQDRLRGKFGDPDVFHWLAEDGQEALWKERDCVTSFSLLNDADGLLEKRKGYALMTFADARLHPRWAMYEAPGGSNLRTYQIRKTENGLWADVVLLSPRNESAAVEEKTFNVRLAPSGQLSNVSFDQIQKGSKMVGRCRYQSANQQFEGLLGGAEILFDRKRIANEQHGATDLASKPGHVWFKLTLDVRPQAPQGWLDGKGRPALPPEAKHFKTALSNKSKFADQVRPGLRVLSVDLGVRSFAACSVFELVRGGPDQGTYFPAADGRTVDDPEKLWAKHERSFKITLPGENPSRKEEIARRAAMEELRSLNGDIRRLKAILRLSVLQEDDPRTEHLRLFMEAIVDDPAKSALNAELFKGFGDDRFRSTPDLWKQHCHFFHDKAEKVVAERFSRWRTETRPKSSSWQDWRERRGYAGGKSYWAVTYLEAVRGLILRWNMRGRTYGEVNRQDKKQFGTVASALLHHINQLKEDRIKTGADMIIQAARGFVPRKNGAGWVQVHEPCRLILFEDLARYRFRTDRSRRENSRLMRWSHREIVNEVGMQGELYGLHVDTTEAGFSSRYLASSGAPGVRCRHLVEEDFHDGLPGMHLVGELDWLLPKDKDRTANEARRLLGGMVRPGMLVPWDGGELFATLNAASQLHVIHADINAAQNLQRRFWGRCGEAIRIVCNQLSVDGSTRYEMAKAPKARLLGALQQLKNGDAPFHLTSIPNSQKPENSYVMTPTNAGKKYRAGPGEKSSGEEDELALDIVEQAEELAQGRKTFFRDPSGVFFAPDRWLPSEIYWSRIRRRIWQVTLERNSSGRQERAEMDEMPY (SEQ ID NO: 73)

The prime editors described herein may also comprise Cas12a (Cpf1)(dCpf1) variants that may be used as a guide nucleotidesequence-programmable DNA-binding protein domain. The Cas12a (Cpf1)protein has a RuvC-like endonuclease domain that is similar to the RuvCdomain of Cas9 but does not have a HNH endonuclease domain, and theN-terminal of Cas12a (Cpf1) does not have the alfa-helical recognitionlobe of Cas9. It was shown in Zetsche et al., Cell, 163, 759-771, 2015(which is incorporated herein by reference) that, the RuvC-like domainof Cas12a (Cpf1) is responsible for cleaving both DNA strands andinactivation of the RuvC-like domain inactivates Cas12a (Cpf1) nucleaseactivity.

In some embodiments, the napDNAbp is a single effector of a microbialCRISPR-Cas system. Single effectors of microbial CRISPR-Cas systemsinclude, without limitation, Cas9, Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a(C2c2), and Cas12c (C2c3). Typically, microbial CRISPR-Cas systems aredivided into Class 1 and Class 2 systems. Class 1 systems havemultisubunit effector complexes, while Class 2 systems have a singleprotein effector. For example, Cas9 and Cas12a (Cpf1) are Class 2effectors. In addition to Cas9 and Cas12a (Cpf1), three distinct Class 2CRISPR-Cas systems (Cas12b1, Cas13a, and Cas12c) have been described byShmakov et al., “Discovery and Functional Characterization of DiverseClass 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, theentire contents of which are hereby incorporated by reference.

Effectors of two of the systems, Cas12b1 and Cas12c, contain RuvC-likeendonuclease domains related to Cas12a. A third system, Cas13a containsan effector with two predicated HEPN RNase domains. Production of matureCRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA byCas12b1. Cas12b1 depends on both CRISPR RNA and tracrRNA for DNAcleavage. Bacterial Cas13a has been shown to possess a unique RNaseactivity for CRISPR RNA maturation distinct from its RNA-activatedsingle-stranded RNA degradation activity. These RNase functions aredifferent from each other and from the CRISPR RNA-processing behavior ofCas12a. See, e.g., East-Seletsky, et al., “Two distinct RNase activitiesof CRISPR-Cas13a enable guide-RNA processing and RNA detection”, Nature,2016 Oct. 13; 538(7624):270-273, the entire contents of which are herebyincorporated by reference. In vitro biochemical analysis of Cas13a inLeptotrichia shahii has shown that Cas13a is guided by a single CRISPRRNA and can be programed to cleave ssRNA targets carrying complementaryprotospacers. Catalytic residues in the two conserved HEPN domainsmediate cleavage. Mutations in the catalytic residues generatecatalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al.,“C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPReffector”, Science, 2016 Aug. 5; 353(6299), the entire contents of whichare hereby incorporated by reference.

The crystal structure of Alicyclobacillus acidoterrestris Cas12b1(AacC2c1) has been reported in complex with a chimeric single-moleculeguide RNA (sgRNA). See e.g., Liu et al., “C2c1-sgRNA Complex StructureReveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19;65(2):310-322, the entire contents of which are hereby incorporated byreference. The crystal structure has also been reported inAlicyclobacillus acidoterrestris C2c1 bound to target DNAs as ternarycomplexes. See e.g., Yang et al., “PAM-dependent Target DNA Recognitionand Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec. 15;167(7):1814-1828, the entire contents of which are hereby incorporatedby reference. Catalytically competent conformations of AacC2c1, bothwith target and non-target DNA strands, have been captured independentlypositioned within a single RuvC catalytic pocket, with C2c1-mediatedcleavage resulting in a staggered seven-nucleotide break of target DNA.Structural comparisons between C2c1 ternary complexes and previouslyidentified Cas9 and Cpf1 counterparts demonstrate the diversity ofmechanisms used by CRISPR-Cas9 systems.

In some embodiments, the napDNAbp may be a C2c1, a C2c2, or a C2c3protein. In some embodiments, the napDNAbp is a C2c1 protein. In someembodiments, the napDNAbp is a Cas13a protein. In some embodiments, thenapDNAbp is a Cas12c protein. In some embodiments, the napDNAbpcomprises an amino acid sequence that is at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to a naturally-occurring Cas12b1 (C2c1), Cas13a (C2c2), orCas12c (C2c3) protein. In some embodiments, the napDNAbp is anaturally-occurring Cas12b1 (C2c1), Cas13a (C2c2), or Cas12c (C2c3)protein.

H. Cas9 Circular Permutants

In various embodiments, the prime editors disclosed herein may comprisea circular permutant of Cas9.

The term “circularly permuted Cas9” or “circular permutant” of Cas9 or“CP-Cas9”) refers to any Cas9 protein, or variant thereof, that occursor has been modify to engineered as a circular permutant variant, whichmeans the N-terminus and the C-terminus of a Cas9 protein (e.g., a wildtype Cas9 protein) have been topically rearranged. Such circularlypermuted Cas9 proteins, or variants thereof, retain the ability to bindDNA when complexed with a guide RNA (gRNA). See, Oakes et al., “ProteinEngineering of Cas9 for enhanced function,” Methods Enzymol, 2014, 546:491-511 and Oakes et al., “CRISPR-Cas9 Circular Permutants asProgrammable Scaffolds for Genome Modification,” Cell, Jan. 10, 2019,176: 254-267, each of are incorporated herein by reference. The instantdisclosure contemplates any previously known CP-Cas9 or use a newCP-Cas9 so long as the resulting circularly permuted protein retains theability to bind DNA when complexed with a guide RNA (gRNA).

Any of the Cas9 proteins described herein, including any variant,ortholog, or naturally occurring Cas9 or equivalent thereof, may bereconfigured as a circular permutant variant.

In various embodiments, the circular permutants of Cas9 may have thefollowing structure: N-terminus-[original C-terminus]-[optionallinker]-[original N-terminus]-C-terminus.

As an example, the present disclosure contemplates the followingcircular permutants of canonical S. pyogenes Cas9 (1368 amino acids ofUniProtKB—Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acidposition in SEQ ID NO: 18)): N-terminus-[1268-1368]-[optionallinker]-[1-1267]-C-terminus; N-terminus-[1168-1368]-[optionallinker]-[1-1167]-C-terminus; N-terminus-[1068-1368]-[optionallinker]-[1-1067]-C-terminus; N-terminus-[968-1368]-[optionallinker]-[1-967]-C-terminus; N-terminus-[868-1368]-[optionallinker]-[1-867]-C-terminus; N-terminus-[768-1368]-[optionallinker]-[1-767]-C-terminus; N-terminus-[668-1368]-[optionallinker]-[1-667]-C-terminus; N-terminus-[568-1368]-[optionallinker]-[1-567]-C-terminus; N-terminus-[468-1368]-[optionallinker]-[1-467]-C-terminus; N-terminus-[368-1368]-[optionallinker]-[1-367]-C-terminus; N-terminus-[268-1368]-[optionallinker]-[1-267]-C-terminus; N-terminus-[168-1368]-[optionallinker]-[1-167]-C-terminus; N-terminus-[68-1368]-[optionallinker]-[1-67]-C-terminus; or N-terminus-[10-1368]-[optionallinker]-[1-9]-C-terminus, or the corresponding circular permutants ofother Cas9 proteins (including other Cas9 orthologs, variants, etc).

In particular embodiments, the circular permutant Cas9 has the followingstructure (based on S. pyogenes Cas9 (1368 amino acids ofUniProtKB—Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acidposition in SEQ ID NO: 18): N-terminus-[102-1368]-[optionallinker]-[1-101]-C-terminus; N-terminus-[1028-1368]-[optionallinker]-[1-1027]-C-terminus; N-terminus-[1041-1368]-[optionallinker]-[1-1043]-C-terminus; N-terminus-[1249-1368]-[optionallinker]-[1-1248]-C-terminus; or N-terminus-[1300-1368]-[optionallinker]-[1-1299]-C-terminus, or the corresponding circular permutants ofother Cas9 proteins (including other Cas9 orthologs, variants, etc).

In still other embodiments, the circular permutant Cas9 has thefollowing structure (based on S. pyogenes Cas9 (1368 amino acids ofUniProtKB—Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acidposition in SEQ ID NO: 18): N-terminus-[103-1368]-[optionallinker]-[1-102]-C-terminus; N-terminus-[1029-1368]-[optionallinker]-[1-1028]-C-terminus; N-terminus-[1042-1368]-[optionallinker]-[1-1041]-C-terminus; N-terminus-[1250-1368]-[optionallinker]-[1-1249]-C-terminus; or N-terminus-[1301-1368]-[optionallinker]-[1-1300]-C-terminus, or the corresponding circular permutants ofother Cas9 proteins (including other Cas9 orthologs, variants, etc).

In some embodiments, the circular permutant can be formed by linking aC-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9,either directly or by using a linker, such as an amino acid linker. Insome embodiments, The C-terminal fragment may correspond to theC-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acidsabout 1300-1368), or the C-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%,55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of aCas9 (e.g., any one of SEQ ID NOs: 77-86). The N-terminal portion maycorrespond to the N-terminal 95% or more of the amino acids of a Cas9(e.g., amino acids about 1-1300), or the N-terminal 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%or more of a Cas9 (e.g., of SEQ ID NO: 18).

In some embodiments, the circular permutant can be formed by linking aC-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9,either directly or by using a linker, such as an amino acid linker. Insome embodiments, the C-terminal fragment that is rearranged to theN-terminus, includes or corresponds to the C-terminal 30% or less of theamino acids of a Cas9 (e.g., amino acids 1012-1368 of SEQ ID NO: 18). Insome embodiments, the C-terminal fragment that is rearranged to theN-terminus, includes or corresponds to the C-terminal 30%, 29%, 28%,27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the aminoacids of a Cas9 (e.g., the Cas9 of SEQ ID NO: 18). In some embodiments,the C-terminal fragment that is rearranged to the N-terminus, includesor corresponds to the C-terminal 410 residues or less of a Cas9 (e.g.,the Cas9 of SEQ ID NO: 18). In some embodiments, the C-terminal portionthat is rearranged to the N-terminus, includes or corresponds to theC-terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300,290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160,150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 18). In someembodiments, the C-terminal portion that is rearranged to theN-terminus, includes or corresponds to the C-terminal 357, 341, 328,120, or 69 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 18).

In other embodiments, circular permutant Cas9 variants may be defined asa topological rearrangement of a Cas9 primary structure based on thefollowing method, which is based on S. pyogenes Cas9 of SEQ ID NO: 18:(a) selecting a circular permutant (CP) site corresponding to aninternal amino acid residue of the Cas9 primary structure, whichdissects the original protein into two halves: an N-terminal region anda C-terminal region; (b) modifying the Cas9 protein sequence (e.g., bygenetic engineering techniques) by moving the original C-terminal region(comprising the CP site amino acid) to preceed the original N-terminalregion, thereby forming a new N-terminus of the Cas9 protein that nowbegins with the CP site amino acid residue. The CP site can be locatedin any domain of the Cas9 protein, including, for example, thehelical-II domain, the RuvCIII domain, or the CTD domain. For example,the CP site may be located (relative the S. pyogenes Cas9 of SEQ ID NO:18) at original amino acid residue 181, 199, 230, 270, 310, 1010, 1016,1023, 1029, 1041, 1247, 1249, or 1282. Thus, once relocated to theN-terminus, original amino acid 181, 199, 230, 270, 310, 1010, 1016,1023, 1029, 1041, 1247, 1249, or 1282 would become the new N-terminalamino acid. Nomenclature of these CP-Cas9 proteins may be referred to asCas9-CP¹⁸¹, Cas9-CP¹⁹⁹, Cas9-CP²³⁰, Cas9-CP²⁷⁰, Cas9-CP³¹⁰, Cas9-CP¹⁰¹⁰,Cas9-CP¹⁰¹⁶, Cas9-CP¹⁰²³, Cas9-CP¹⁰²⁹, Cas9-CP¹⁰⁴¹, Cas9-CP¹²⁴⁷,Cas9-CP¹²⁴⁹, and Cas9-CP¹²⁸², respectively. This description is notmeant to be limited to making CP variants from SEQ ID NO: 18, but may beimplemented to make CP variants in any Cas9 sequence, either at CP sitesthat correspond to these positions, or at other CP sites entirely. Thisdescription is not meant to limit the specific CP sites in any way.Virtually any CP site may be used to form a CP-Cas9 variant.

Exemplary CP-Cas9 amino acid sequences, based on the Cas9 of SEQ ID NO:18, are provided below in which linker sequences are indicated byunderlining and optional methionine (M) residues are indicated in bold.It should be appreciated that the disclosure provides CP-Cas9 sequencesthat do not include a linker sequence or that include different linkersequences. It should be appreciated that CP-Cas9 sequences may be basedon Cas9 sequences other than that of SEQ ID NO: 18 and any examplesprovided herein are not meant to be limiting. Exemplary CP-Cas9sequences are as follows:

CP name Sequence SEQ ID NO: CP1012DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE SEQ ID NO:ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS 77MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYG CP1028EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG SEQ ID NO:ETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG 78FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGG SGGSGGSGGSGGSGGMDKKYSIGLAIGTNSVGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF VYGDYKVYDVRKMIAKSEQ CP1041NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD SEQ ID NO:FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSD 79KLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEQEIGKATAKYFFYS CP1249PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL SEQ ID NO:DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFK 80YFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGDGGSGGSGGSGGSGGSGGSGGMDKKYSIGLAIGTN SVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGS CP1300KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTST SEQ ID NO:KEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGS 81GGSGGSGGSGGDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR VILADANLDKVLSAYNKHRD

The Cas9 circular permutants that may be useful in the prime editingconstructs described herein. Exemplary C-terminal fragments of Cas9,based on the Cas9 of SEQ ID NO: 18, which may be rearranged to anN-terminus of Cas9, are provided below. It should be appreciated thatsuch C-terminal fragments of Cas9 are exemplary and are not meant to belimiting. These exemplary CP-Cas9 fragments have the followingsequences:

CP name Sequence SEQ ID NO: CP1012 C-DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT SEQ ID NO: terminalEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL 82 fragmentSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGL YETRIDLSQLGGD CP1028 C-EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG SEQ ID NO: terminalETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG 83 fragmentFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD CP1041 C-NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD SEQ ID NO: terminalFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSD 84 fragmentKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGDCP1249 C- PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL SEQ ID NO: terminalDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFK 85 fragmentYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGD CP1300 C-KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTST SEQ ID NO: terminalKEVLDATLIHQSITGLYETRIDLSQLGGD 86 fragment

I. Cas9 Variants with Modified PAM Specificities

The prime editors of the present disclosure may also comprise Cas9variants with modified PAM specificities. Some aspects of thisdisclosure provide Cas9 proteins that exhibit activity on a targetsequence that does not comprise the canonical PAM (5′-NGG-3′, where N isA, C, G, or T) at its 3′-end. In some embodiments, the Cas9 proteinexhibits activity on a target sequence comprising a 5′-NGG-3′ PAMsequence at its 3′-end. In some embodiments, the Cas9 protein exhibitsactivity on a target sequence comprising a 5′-NNG-3′ PAM sequence at its3′-end. In some embodiments, the Cas9 protein exhibits activity on atarget sequence comprising a 5′-NNA-3′ PAM sequence at its 3′-end. Insome embodiments, the Cas9 protein exhibits activity on a targetsequence comprising a 5′-NNC-3′ PAM sequence at its 3′-end. In someembodiments, the Cas9 protein exhibits activity on a target sequencecomprising a 5′-NNT-3′ PAM sequence at its 3′-end. In some embodiments,the Cas9 protein exhibits activity on a target sequence comprising a5′-NGT-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9protein exhibits activity on a target sequence comprising a 5′-NGA-3′PAM sequence at its 3′-end. In some embodiments, the Cas9 proteinexhibits activity on a target sequence comprising a 5′-NGC-3′ PAMsequence at its 3′-end. In some embodiments, the Cas9 protein exhibitsactivity on a target sequence comprising a 5′-NAA-3′ PAM sequence at its3′-end. In some embodiments, the Cas9 protein exhibits activity on atarget sequence comprising a 5′-NAC-3′ PAM sequence at its 3′-end. Insome embodiments, the Cas9 protein exhibits activity on a targetsequence comprising a 5′-NAT-3′ PAM sequence at its 3′-end. In stillother embodiments, the Cas9 protein exhibits activity on a targetsequence comprising a 5′-NAG-3′ PAM sequence at its 3′-end.

It should be appreciated that any of the amino acid mutations describedherein, (e.g., A262T) from a first amino acid residue (e.g., A) to asecond amino acid residue (e.g., T) may also include mutations from thefirst amino acid residue to an amino acid residue that is similar to(e.g., conserved) the second amino acid residue. For example, mutationof an amino acid with a hydrophobic side chain (e.g., alanine, valine,isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan)may be a mutation to a second amino acid with a different hydrophobicside chain (e.g., alanine, valine, isoleucine, leucine, methionine,phenylalanine, tyrosine, or tryptophan). For example, a mutation of analanine to a threonine (e.g., a A262T mutation) may also be a mutationfrom an alanine to an amino acid that is similar in size and chemicalproperties to a threonine, for example, serine. As another example,mutation of an amino acid with a positively charged side chain (e.g.,arginine, histidine, or lysine) may be a mutation to a second amino acidwith a different positively charged side chain (e.g., arginine,histidine, or lysine). As another example, mutation of an amino acidwith a polar side chain (e.g., serine, threonine, asparagine, orglutamine) may be a mutation to a second amino acid with a differentpolar side chain (e.g., serine, threonine, asparagine, or glutamine).Additional similar amino acid pairs include, but are not limited to, thefollowing: phenylalanine and tyrosine; asparagine and glutamine;methionine and cysteine; aspartic acid and glutamic acid; and arginineand lysine. The skilled artisan would recognize that such conservativeamino acid substitutions will likely have minor effects on proteinstructure and are likely to be well tolerated without compromisingfunction. In some embodiments, any amino of the amino acid mutationsprovided herein from one amino acid to a threonine may be an amino acidmutation to a serine. In some embodiments, any amino of the amino acidmutations provided herein from one amino acid to an arginine may be anamino acid mutation to a lysine. In some embodiments, any amino of theamino acid mutations provided herein from one amino acid to anisoleucine, may be an amino acid mutation to an alanine, valine,methionine, or leucine. In some embodiments, any amino of the amino acidmutations provided herein from one amino acid to a lysine may be anamino acid mutation to an arginine. In some embodiments, any amino ofthe amino acid mutations provided herein from one amino acid to anaspartic acid may be an amino acid mutation to a glutamic acid orasparagine. In some embodiments, any amino of the amino acid mutationsprovided herein from one amino acid to a valine may be an amino acidmutation to an alanine, isoleucine, methionine, or leucine. In someembodiments, any amino of the amino acid mutations provided herein fromone amino acid to a glycine may be an amino acid mutation to an alanine.It should be appreciated, however, that additional conserved amino acidresidues would be recognized by the skilled artisan and any of the aminoacid mutations to other conserved amino acid residues are also withinthe scope of this disclosure.

In some embodiments, the Cas9 protein comprises a combination ofmutations that exhibit activity on a target sequence comprising a5′-NAA-3′ PAM sequence at its 3′-end. In some embodiments, thecombination of mutations are present in any one of the clones listed inTable 1. In some embodiments, the combination of mutations areconservative mutations of the clones listed in Table 1. In someembodiments, the Cas9 protein comprises the combination of mutations ofany one of the Cas9 clones listed in Table 1.

TABLE 1 NAA PAM Clones Mutations from wild-type SpCas9 (e.g., SEQ ID NO:18) D177N, K218R, D614N, D1135N, P1137S, E1219V, A1320V, A1323D, R1333KD177N, K218R, D614N, D1135N, E1219V, Q1221H, H1264Y, A1320V, R1333KA10T, I322V, S409I, E427G, G715C, D1135N, E1219V, Q1221H, H1264Y,A1320V, R1333K A367T, K710E, R1114G, D1135N, P1137S, E1219V, Q1221H,H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, R753G, D861N, D1135N,K1188R, E1219V, Q1221H, H1264H, A1320V, R1333K A10T, I322V, S409I,E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G, K1211R, E1219V,Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, V743I, R753G,E762G, D1135N, D1180G, K1211R, E1219V, Q1221H, H1264Y, A1320V, R1333KA10T, I322V, S409I, E427G, R753G, D1135N, D1180G, K1211R, E1219V,Q1221H, H1264Y, S1274R, A1320V, R1333K A10T, I322V, S409I, E427G, A5895,R753G, D1135N, E1219V, Q1221H, H1264H, A1320V, R1333K A10T, I322V,S409I, E427G, R753G, E757K, G865G, D1135N, E1219V, Q1221H, H1264Y,A1320V, R1333K A10T, I322V, S409I, E427G, R654L, R753G, E757K, D1135N,E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, K599R,M631A, R654L, K673E, V743I, R753G, N758H, E762G, D1135N, D1180G, E1219V,Q1221H, Q1256R, H1264Y, A1320V, A1323D, R1333K A10T, I322V, S409I,E427G, R654L, K673E, V743I, R753G, E762G, N8695, N1054D, R1114G, D1135N,D1180G, E1219V, Q1221H, H1264Y, A1320V, A1323D, R1333K A10T, I322V,S409I, E427G, R654L, L727I, V743I, R753G, E762G, R8595, N946D, F1134L,D1135N, D1180G, E1219V, Q1221H, H1264Y, N1317T, A1320V, A1323D, R1333KA10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S,N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H,H1264Y, V1290G, L13185, A1320V, A1323D, R1333K A10T, I322V, S409I,E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S, Y1016D, G1077D,R1114G, F1134L, D1135N, K1151E, D1180G, E1219V, Q1221H, H1264Y, V1290G,L13185, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E, V743I,R753G, E762G, N803S, N8695, Y1016D, G1077D, R1114G, F1134L, D1135N,D1180G, E1219V, Q1221H, H1264Y, V1290G, L1318S, A1320V, A1323D, R1333KA10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G,N803S, N869S, L921P, Y1016D, G1077D, F1080S, R1114G, D1135N, D1180G,E1219V, Q1221H, H1264Y, L1318S, A1320V, A1323D, R1333K A10T, I322V,S409I, E427G, E630K, R654L, K673E, V743I, R753G, E762G, Q768H, N803S,N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H,H1264Y, L1318S, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E,F693L, V743I, R753G, E762G, Q768H, N803S, N869S, Y1016D, G1077D, R1114G,F1134L, D1135N, D1180G, E1219V, Q1221H, G1223S, H1264Y, L1318S, A1320V,R1333K A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G,E762G, N803S, N869S, L921P, Y1016D, G1077D, F1801S, R1114G, D1135N,D1180G, E1219V, Q1221H, H1264Y, L1318S, A1320V, A1323D, R1333K A10T,I322V, S409I, E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G,K1211R, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I,E427G, R654L, K673E, V743I, R753G, E762G, M673I, N803S, N869S, G1077D,R1114G, D1135N, V1139A, D1180G, E1219V, Q1221H, A1320V, R1333K A10T,I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,R1114G, D1135N, E1219V, Q1221H, A1320V, R1333K

In some embodiments, the Cas9 protein comprises an amino acid sequencethat is at least 80% identical to the amino acid sequence of a Cas9protein as provided by any one of the variants of Table 1. In someembodiments, the Cas9 protein comprises an amino acid sequence that isat least 85%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence of a Cas9 protein as provided by any one of thevariants of Table 1.

In some embodiments, the Cas9 protein exhibits an increased activity ona target sequence that does not comprise the canonical PAM (5′-NGG-3′)at its 3′ end as compared to Streptococcus pyogenes Cas9 as provided bySEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits anactivity on a target sequence having a 3′ end that is not directlyadjacent to the canonical PAM sequence (5′-NGG-3′) that is at least5-fold increased as compared to the activity of Streptococcus pyogenesCas9 as provided by SEQ ID NO: 18 on the same target sequence. In someembodiments, the Cas9 protein exhibits an activity on a target sequencethat is not directly adjacent to the canonical PAM sequence (5′-NGG-3′)that is at least 10-fold, at least 50-fold, at least 100-fold, at least500-fold, at least 1,000-fold, at least 5,000-fold, at least10,000-fold, at least 50,000-fold, at least 100,000-fold, at least500,000-fold, or at least 1,000,000-fold increased as compared to theactivity of Streptococcus pyogenes as provided by SEQ ID NO: 18 on thesame target sequence. In some embodiments, the 3′ end of the targetsequence is directly adjacent to an AAA, GAA, CAA, or TAA sequence. Insome embodiments, the Cas9 protein comprises a combination of mutationsthat exhibit activity on a target sequence comprising a 5′-NAC-3′ PAMsequence at its 3′-end. In some embodiments, the combination ofmutations are present in any one of the clones listed in Table 2. Insome embodiments, the combination of mutations are conservativemutations of the clones listed in Table 2. In some embodiments, the Cas9protein comprises the combination of mutations of any one of the Cas9clones listed in Table 2.

TABLE 2 NAC PAM Clones MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO:18) T472I, R753G, K890E, D1332N, R1335Q, T1337N I1057S, D1135N, P1301S,R1335Q, T1337N T472I, R753G, D1332N, R1335Q, T1337N D1135N, E1219V,D1332N, R1335Q, T1337N T472I, R753G, K890E, D1332N, R1335Q, T1337NI1057S, D1135N, P1301S, R1335Q, T1337N T472I, R753G, D1332N, R1335Q,T1337N T472I, R753G, Q771H, D1332N, R1335Q, T1337N E627K, T638P, K652T,R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337NE627K, T638P, K652T, R753G, N803S, K959N, R1114G, D1135N, K1156E,E1219V, D1332N, R1335Q, T1337N E627K, T638P, V647I, R753G, N803S, K959N,G1030R, I1055E, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N E627K,E630G, T638P, V647A, G687R, N767D, N803S, K959N, R1114G, D1135N, E1219V,D1332G, R1335Q, T1337N E627K, T638P, R753G, N803S, K959N, R1114G,D1135N, E1219V, N1266H, D1332N, R1335Q, T1337N E627K, T638P, R753G,N803S, K959N, I1057T, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337NE627K, T638P, R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N,R1335Q, T1337N E627K, M631I, T638P, R753G, N803S, K959N, Y1036H, R1114G,D1135N, E1219V, D1251G, D1332G, R1335Q, T1337N E627K, T638P, R753G,N803S, V875I, K959N, Y1016C, R1114G, D1135N, E1219V, D1251G, D1332G,R1335Q, T1337N, I1348V K608R, E627K, T638P, V647I, R654L, R753G, N803S,T804A, K848N, V922A, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q,T1337N K608R, E627K, T638P, V647I, R753G, N803S, V922A, K959N, K1014N,V1015A, R1114G, D1135N, K1156N, E1219V, N1252D, D1332N, R1335Q, T1337NK608R, E627K, R629G, T638P, V647I, A711T, R753G, K775R, K789E, N803S,K959N, V1015A, Y1036H, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q,T1337N K608R, E627K, T638P, V647I, T740A, R753G, N803S, K948E, K959N,Y1016S, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N K608R,E627K, T638P, V647I, T740A, N803S, K948E, K959N, Y1016S, R1114G, D1135N,E1219V, N1286H, D1332N, R1335Q, T1337N I670S, K608R, E627K, E630G,T638P, V647I, R653K, R753G, I795L, K797N, N803S, K866R, K890N, K959N,Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N K608R, E627K,T638P, V647I, T740A, G752R, R753G, K797N, N803S, K948E, K959N, V1015A,Y1016S, R1114G, D1135N, E1219V, N1266H, D1332N, R1335Q, T1337N I570T,A589V, K608R, E627K, T638P, V647I, R654L, Q716R, R753G, N803S, K948E,K959N, Y1016S, R1114G, D1135N, E1207G, E1219V, N1234D, D1332N, R1335Q,T1337N K608R, E627K, R629G, T638P, V647I, R654L, Q740R, R753G, N803S,K959N, N990S, T995S, V1015A, Y1036D, R1114G, D1135N, E1207G, E1219V,N1234D, N1266H, D1332N, R1335Q, T1337N I562F, V565D, I570T, K608R,L625S, E627K, T638P, V647I, R654I, G752R, R753G, N803S, N808D, K959N,M1021L, R1114G, D1135N, N1177S, N1234D, D1332N, R1335Q, T1337N I562F,I570T, K608R, E627K, T638P, V647I, R753G, E790A, N803S, K959N, V1015A,Y1036H, R1114G, D1135N, D1180E, A1184T, E1219V, D1332N, R1335Q, T1337NI570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N,V1015A, R1114G, D1127A, D1135N, E1219V, D1332N, R1335Q, T1337N I570T,K608R, L625S, E627K, T638P, V647I, R654I, T703P, R753G, N803S, N808D,K959N, M1021L, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N I570S,K608R, E627K, E630G, T638P, V647I, R653K, R753G, I795L, N803S, K866R,K890N, K959N, Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337NI570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N,V1016A, R1114G, D1135N, E1219V, K1246E, D1332N, R1335Q, T1337N K608R,E627K, T638P, V647I, R654L, K673E, R753G, E790A, N803S, K948E, K959N,R1114G, D1127G, D1135N, D1180E, E1219V, N1286H, D1332N, R1335Q, T1337NK608R, L625S, E627K, T638P, V647I, R654I, I670T, R753G, N803S, N808D,K959N, M1021L, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337NE627K, M631V, T638P, V647I, K710E, R753G, N803S, N808D, K948E, M1021L,R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N, S1338T, H1349R

In some embodiments, the Cas9 protein comprises an amino acid sequencethat is at least 80% identical to the amino acid sequence of a Cas9protein as provided by any one of the variants of Table 2. In someembodiments, the Cas9 protein comprises an amino acid sequence that isat least 85%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence of a Cas9 protein as provided by any one of thevariants of Table 2.

In some embodiments, the Cas9 protein exhibits an increased activity ona target sequence that does not comprise the canonical PAM (5′-NGG-3′)at its 3′ end as compared to Streptococcus pyogenes Cas9 as provided bySEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits anactivity on a target sequence having a 3′ end that is not directlyadjacent to the canonical PAM sequence (5′-NGG-3′) that is at least5-fold increased as compared to the activity of Streptococcus pyogenesCas9 as provided by SEQ ID NO: 18 on the same target sequence. In someembodiments, the Cas9 protein exhibits an activity on a target sequencethat is not directly adjacent to the canonical PAM sequence (5′-NGG-3′)that is at least 10-fold, at least 50-fold, at least 100-fold, at least500-fold, at least 1,000-fold, at least 5,000-fold, at least10,000-fold, at least 50,000-fold, at least 100,000-fold, at least500,000-fold, or at least 1,000,000-fold increased as compared to theactivity of Streptococcus pyogenes as provided by SEQ ID NO: 18 on thesame target sequence. In some embodiments, the 3 end of the targetsequence is directly adjacent to an AAC, GAC, CAC, or TAC sequence.

In some embodiments, the Cas9 protein comprises a combination ofmutations that exhibit activity on a target sequence comprising a5′-NAT-3′ PAM sequence at its 3′-end. In some embodiments, thecombination of mutations are present in any one of the clones listed inTable 3. In some embodiments, the combination of mutations areconservative mutations of the clones listed in Table 3. In someembodiments, the Cas9 protein comprises the combination of mutations ofany one of the Cas9 clones listed in Table 3.

TABLE 3 NAT PAM Clones MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO:18) K961E, H985Y, D1135N, K1191N, E1219V, Q1221H, A1320A, P1321S, R1335LD1135N, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L V743I,R753G, E790A, D1135N, G1218S, E1219V, Q1221H, A1227V, P1249S, N1286K,A1293T, P1321S, D1322G, R1335L, T1339I F575S, M631L, R654L, V748I,V743I, R753G, D853E, V922A, R1114G D1135N, G1218S, E1219V, Q1221H,A1227V, P1249S, N1286K, A1293T, P1321S, D1322G, R1335L, T1339I F575S,M631L, R654L, R664K, R753G, D853E, V922A, R1114G D1135N, D1180G, G1218S,E1219V, Q1221H, P1249S, N1286K, P1321S, D1322G, R1335L M631L, R654L,R753G, K797E, D853E, V922A, D1012A, R1114G D1135N, G1218S, E1219V,Q1221H, P1249S, N1317K, P1321S, D1322G, R1335L F575S, M631L, R654L,R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S,E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, M631L, R654L,R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S,E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, D596Y, M631L,R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G,G1218S, E1219V, Q1221H, P1249S, Q1256R, P1321S, D1322G, R1335L F575S,M631L, R654L, R664K, K710E, V750A, R753G, D853E, V922A, R1114G, Y1131C,D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335LF575S, M631L, K649R, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C,D1135N, K1156E D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G,R1335L F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C,D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335LF575S, M631L, R654L, R664K, R753G, D853E, V922A, I1057G, R1114G, Y1131C,D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, N1308D, P1321S, D1322G,R1335L M631L, R654L, R753G, D853E, V922A, R1114G, Y1131C, D1135N,E1150V, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335LM631L, R654L, R664K, R753G, D853E, I1057V, Y1131C, D1135N, D1180G,G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L M631L, R654L,R664K, R753G, I1057V, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V,Q1221H, P1249S, P1321S, D1332G, R1335L

The above description of various napDNAbps which can be used inconnection with the presently disclose prime editors is not meant to belimiting in any way. The prime editors may comprise the canonicalSpCas9, or any ortholog Cas9 protein, or any variant Cas9protein—including any naturally occurring variant, mutant, or otherwiseengineered version of Cas9—that is known or which can be made or evolvedthrough a directed evolutionary or otherwise mutagenic process. Invarious embodiments, the Cas9 or Cas9 variants have a nickase activity,i.e., only cleave of strand of the target DNA sequence. In otherembodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e.,are “dead” Cas9 proteins. Other variant Cas9 proteins that may be usedare those having a smaller molecular weight than the canonical SpCas9(e.g., for easier delivery) or having modified or rearranged primaryamino acid structure (e.g., the circular permutant formats). The primeeditors described herein may also comprise Cas9 equivalents, includingCas12a/Cpf1 and Cas12b proteins which are the result of convergentevolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, orCas9 equivalents) may also may also contain various modifications thatalter/enhance their PAM specifities. Lastly, the applicationcontemplates any Cas9, Cas9 variant, or Cas9 equivalent which has atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.9%sequence identity to a reference Cas9 sequence, such as a referencesSpCas9 canonical sequences or a reference Cas9 equivalent (e.g.,Cas12a/Cpf1).

In a particular embodiment, the Cas9 variant having expanded PAMcapabilities is SpCas9 (H840A) VRQR (SEQ ID NO: 87), which has thefollowing amino acid sequence (with the V, R, Q, R substitutionsrelative to the SpCas9 (H840A) of SEQ ID NO: 51 being show in boldunderline. In addition, the methionine residue in SpCas9 (H840) wasremoved for SpCas9 (H840A) VRQR):

(SEQ ID NO: 87) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASA RELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR Q Y R STKEVLDATLIHQSI TGLYETRIDLSQLGGD

In another particular embodiment, the Cas9 variant having expanded PAMcapabilities is SpCas9 (H840A) VRER, which has the following amino acidsequence (with the V, R, E, R substitutions relative to the SpCas9(H840A) of SEQ ID NO: 51 being shown in bold underline. In addition, themethionine residue in SpCas9 (H840) was removed for SpCas9 (H840A)VRER):

(SEQ ID NO: 88) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASA RELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK E Y R STKEVLDATLIHQS ITGLYETRIDLSQLGGD

In some embodiments, the napDNAbp that functions with a non-canonicalPAM sequence is an Argonaute protein. One example of such a nucleic acidprogrammable DNA binding protein is an Argonaute protein fromNatronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease.NgAgo binds 5′ phosphorylated ssDNA of ˜24 nucleotides (gDNA) to guideit to its target site and will make DNA double-strand breaks at the gDNAsite. In contrast to Cas9, the NgAgo-gDNA system does not require aprotospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo(dNgAgo) can greatly expand the bases that may be targeted. Thecharacterization and use of NgAgo have been described in Gao et al., NatBiotechnol., 2016 July; 34(7):768-73. PubMed PMID: 27136078; Swarts etal., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic AcidsRes. 43(10) (2015):5120-9, each of which is incorporated herein byreference.

In some embodiments, the napDNAbp is a prokaryotic homolog of anArgonaute protein. Prokaryotic homologs of Argonaute proteins are knownand have been described, for example, in Makarova K., et al.,“Prokaryotic homologs of Argonaute proteins are predicted to function askey components of a novel system of defense against mobile geneticelements”, Biol Direct. 2009 Aug. 25; 4:29. doi: 10.1186/1745-6150-4-29,the entire contents of which is hereby incorporated by reference. Insome embodiments, the napDNAbp is a Marinitoga piezophila Argunaute(MpAgo) protein. The CRISPR-associated Marinitoga piezophila Argunaute(MpAgo) protein cleaves single-stranded target sequences using5′-phosphorylated guides. The 5′ guides are used by all knownArgonautes. The crystal structure of an MpAgo-RNA complex shows a guidestrand binding site comprising residues that block 5′ phosphateinteractions. This data suggests the evolution of an Argonaute subclasswith noncanonical specificity for a 5′-hydroxylated guide. See, e.g.,Kaya et al., “A bacterial Argonaute with noncanonical guide RNAspecificity”, Proc Natl Acad Sci USA. 2016 Apr. 12; 113(15):4057-62, theentire contents of which are hereby incorporated by reference). Itshould be appreciated that other argonaute proteins may be used, and arewithin the scope of this disclosure.

Some aspects of the disclosure provide Cas9 domains that have differentPAM specificities. Typically, Cas9 proteins, such as Cas9 from S.pyogenes (spCas9), require a canonical NGG PAM sequence to bind aparticular nucleic acid region. This may limit the ability to editdesired bases within a genome. In some embodiments, the base editingfusion proteins provided herein may need to be placed at a preciselocation, for example where a target base is placed within a 4 baseregion (e.g., a “editing window”), which is approximately 15 basesupstream of the PAM. See Komor, A. C., et al., “Programmable editing ofa target base in genomic DNA without double-stranded DNA cleavage”Nature 533, 420-424 (2016), the entire contents of which are herebyincorporated by reference. Accordingly, in some embodiments, any of thefusion proteins provided herein may contain a Cas9 domain that iscapable of binding a nucleotide sequence that does not contain acanonical (e.g., NGG) PAM sequence. Cas9 domains that bind tonon-canonical PAM sequences have been described in the art and would beapparent to the skilled artisan. For example, Cas9 domains that bindnon-canonical PAM sequences have been described in Kleinstiver, B. P.,et al., “Engineered CRISPR-Cas9 nucleases with altered PAMspecificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., etal., “Broadening the targeting range of Staphylococcus aureusCRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33,1293-1298 (2015); the entire contents of each are hereby incorporated byreference.

For example, a napDNAbp domain with altered PAM specificity, such as adomain with at least 80%, at least 85%, at least 90%, at least 95%, orat least 99% sequence identity with wild type Francisella novicida Cpf1(D917, E1006, and D1255) (SEQ ID NO: 74), which has the following aminoacid sequence:

(SEQ ID NO: 74) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

An additional napDNAbp domain with altered PAM specificity, such as adomain having at least 80%, at least 85%, at least 90%, at least 95%, orat least 99% sequence identity with wild type Geobacillusthermodenitrificans Cas9 (SEQ ID NO: 75), which has the following aminoacid sequence:

(SEQ ID NO: 75) MKYKIGLDIGITSIGWAVINLDIPRIEDLGVRIFDRAENPKTGESLALPRRLARSARRRLRRRKHRLERIRRLFVREGILTKEELNKLFEKKHEIDVWQLRVEALDRKLNNDELARILLHLAKRRGFRSNRKSERTNKENSTMLKHIEENQSILSSYRTVAEMVVKDPKFSLHKRNKEDNYTNTVARDDLEREIKLIFAKQREYGNIVCTEAFEHEYISIWASQRPFASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFTVWEHINKLRLVSPGGIRALTDDERRLIYKQAFHKNKITFHDVRTLLNLPDDTRFKGLLYDRNTTLKENEKVRFLELGAYHKIRKAIDSVYGKGAAKSFRPIDFDTFGYALTMFKDDTDIRSYLRNEYEQNGKRMENLADKVYDEELIEELLNLSFSKFGHLSLKALRNILPYMEQGEVYSTACERAGYTFTGPKKKQKTVLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARELSQSFDERRKMQKEQEGNRKKNETAIRQLVEYGLTLNPTGLDIVKFKLWSEQNGKCAYSLQPIEIERLLEPGYTEVDHVIPYSRSLDDSYTNKVLVLTKENREKGNRTPAEYLGLGSERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEENEFKNRNLNDTRYISRFLANFIREHLKFADSDDKQKVYTVNGRITAHLRSRWNFNKNREESNLHHAVDAAIVACTTPSDIARVTAFYQRREQNKELSKKTDPQFPQPWPHFADELQARLSKNPKESIKALNLGNYDNEKLESLQPVFVSRMPKRSITGAAHQETLRRYIGIDERSGKIQTVVKKKLSEIQLDKTGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGELGPIIRTIKIIDTTNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPIYTIDMMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIEFPREKTIKTAVGEEIKIKDLFAYYQTIDSSNGGLSLVSHDNNFSLRSIGSRTLKRFEKYQVDVLGNIYKVRGEKRVGVASSSHSKAGETIRPL

In some embodiments, the nucleic acid programmable DNA binding protein(napDNAbp) is a nucleic acid programmable DNA binding protein that doesnot require a canonical (NGG) PAM sequence. In some embodiments, thenapDNAbp is an argonaute protein. One example of such a nucleic acidprogrammable DNA binding protein is an Argonaute protein fromNatronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease.NgAgo binds 5′ phosphorylated ssDNA of ˜24 nucleotides (gDNA) to guideit to its target site and will make DNA double-strand breaks at the gDNAsite. In contrast to Cas9, the NgAgo-gDNA system does not require aprotospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo(dNgAgo) can greatly expand the bases that may be targeted. Thecharacterization and use of NgAgo have been described in Gao et al., NatBiotechnol., 34(7): 768-73 (2016), PubMed PMID: 27136078; Swarts et al.,Nature, 507(7491): 258-61 (2014); and Swarts et al., Nucleic Acids Res.43(10) (2015): 5120-9, each of which is incorporated herein byreference. The sequence of Natronobacterium gregoryi Argonaute isprovided in SEQ ID NO: 76.

The disclosed fusion proteins may comprise a napDNAbp domain having atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity with wild type Natronobacterium gregoryi Argonaute(SEQ ID NO: 76), which has the following amino acid sequence:

(SEQ ID NO: 76) MTVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPRMSLAFEQDNGERRYITLWKNTTPKDVFTYDYATGSTYIFTNIDYEVKDGYENLTATYQTTVENATAQEVGTTDEDETFAGGEPLDHHLDDALNETPDDAETESDSGHVMTSFASRDQLPEWTLHTYTLTATDGAKTDTEYARRTLAYTVRQELYTDHDAAPVATDGLMLLTPEPLGETPLDLDCGVRVEADETRTLDYTTAKDRLLARELVEEGLKRSLWDDYLVRGIDEVLSKEPVLTCDEFDLHERYDLSVEVGHSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTTYRPRRGHIVWGLRDECATDSLNTLGNQSVVAYHRNNQTPINTDLLDAIEAADRRVVETRRQGHGDDAVSFPQELLAVEPNTHQIKQFASDGFHQQARSKTRLSASRCSEKAQAFAERLDPVRLNGSTVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAHPDETFSKGIVNPPESFEVAVVLPEQQADTCKAQWDTMADLLNQAGAPPTRSETVQYDAFSSPESISLNVAGAIDPSEVDAAFVVLPPDQEGFADLASPTETYDELKKALANMGIYSQMAYFDRFRDAKIFYTRNVALGLLAAAGGVAFTTEHAMPGDADMFIGIDVSRSYPEDGASGQINIAATATAVYKDGTILGHSSTRPQLGEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGFMNEDLDPATEFLNEQGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNEPRATVATFGAPEYLATRDGGGLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHNSTARLPITTAYADQASTHATKGYLVQTGAFESNVGFL

In addition, any available methods may be utilized to obtain orconstruct a variant or mutant Cas9 protein. The term “mutation,” as usedherein, refers to a substitution of a residue within a sequence, e.g., anucleic acid or amino acid sequence, with another residue, or a deletionor insertion of one or more residues within a sequence. Mutations aretypically described herein by identifying the original residue followedby the position of the residue within the sequence and by the identityof the newly substituted residue. Various methods for making the aminoacid substitutions (mutations) provided herein are well known in theart, and are provided by, for example, Green and Sambrook, MolecularCloning: A Laboratory Manual (4th ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2012)). Mutations can include a varietyof categories, such as single base polymorphisms, microduplicationregions, indel, and inversions, and is not meant to be limiting in anyway. Mutations can include “loss-of-function” mutations which is thenormal result of a mutation that reduces or abolishes a proteinactivity. Most loss-of-function mutations are recessive, because in aheterozygote the second chromosome copy carries an unmutated version ofthe gene coding for a fully functional protein whose presencecompensates for the effect of the mutation. Mutations also embrace“gain-of-function” mutations, which is one which confers an abnormalactivity on a protein or cell that is otherwise not present in a normalcondition. Many gain-of-function mutations are in regulatory sequencesrather than in coding regions, and can therefore have a number ofconsequences. For example, a mutation might lead to one or more genesbeing expressed in the wrong tissues, these tissues gaining functionsthat they normally lack. Because of their nature, gain-of-functionmutations are usually dominant.

Mutations can be introduced into a reference Cas9 protein usingsite-directed mutagenesis. Older methods of site-directed mutagenesisknown in the art rely on sub-cloning of the sequence to be mutated intoa vector, such as an M13 bacteriophage vector, that allows the isolationof single-stranded DNA template. In these methods, one anneals amutagenic primer (i.e., a primer capable of annealing to the site to bemutated but bearing one or more mismatched nucleotides at the site to bemutated) to the single-stranded template and then polymerizes thecomplement of the template starting from the 3′ end of the mutagenicprimer. The resulting duplexes are then transformed into host bacteriaand plaques are screened for the desired mutation. More recently,site-directed mutagenesis has employed PCR methodologies, which have theadvantage of not requiring a single-stranded template. In addition,methods have been developed that do not require sub-cloning. Severalissues must be considered when PCR-based site-directed mutagenesis isperformed. First, in these methods it is desirable to reduce the numberof PCR cycles to prevent expansion of undesired mutations introduced bythe polymerase. Second, a selection must be employed in order to reducethe number of non-mutated parental molecules persisting in the reaction.Third, an extended-length PCR method is preferred in order to allow theuse of a single PCR primer set. And fourth, because of thenon-template-dependent terminal extension activity of some thermostablepolymerases it is often necessary to incorporate an end-polishing stepinto the procedure prior to blunt-end ligation of the PCR-generatedmutant product.

Mutations may also be introduced by directed evolution processes, suchas phage-assisted continuous evolution (PACE) or phage-assistednoncontinuous evolution (PANCE). The term “phage-assisted continuousevolution (PACE),” as used herein, refers to continuous evolution thatemploys phage as viral vectors. The general concept of PACE technologyhas been described, for example, in International PCT application,PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 onMar. 11, 2010; International PCT application, PCT/US2011/066747, filedDec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S.application, U.S. Pat. No. 9,023,594, issued May 5, 2015, InternationalPCT application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO2015/134121 on Sep. 11, 2015, and International PCT application,PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 onOct. 20, 2016, the entire contents of each of which are incorporatedherein by reference. Variant Cas9s may also be obtain by phage-assistednon-continuous evolution (PANCE),” which as used herein, refers tonon-continuous evolution that employs phage as viral vectors. PANCE is asimplified technique for rapid in vivo directed evolution using serialflask transfers of evolving ‘selection phage’ (SP), which contain a geneof interest to be evolved, across fresh E. coli host cells, therebyallowing genes inside the host E. coli to be held constant while genescontained in the SP continuously evolve. Serial flask transfers havelong served as a widely-accessible approach for laboratory evolution ofmicrobes, and, more recently, analogous approaches have been developedfor bacteriophage evolution. The PANCE system features lower stringencythan the PACE system.

Any of the references noted above which relate to Cas9 or Cas9equivalents are hereby incorporated by reference in their entireties, ifnot already stated so.

J. Divided napDNAbp Domains for Split PE Delivery

In various embodiments, the prime editors described herein may bedelivered to cells as two or more fragments which become assembledinside the cell (either by passive assembly, or by active assembly, suchas using split intein sequences) into a reconstituted prime editor. Insome cases, the self assembly may be passive whereby the two or moreprime editor fragments associate inside the cell covalently ornon-covalently to reconstitute the prime editor. In other cases, theself-assembly may be catalyzed by dimerization domains installed on eachof the fragments. Examples of dimerization domains are described herein.In still other cases, the self-assembly may be catalyzed by split inteinsequences installed on each of the prime editor fragments.

Split PE delivery may be advantageous to address various sizeconstraints of different delivery approaches. For example, deliveryapproaches may include virus-based delivery methods, messenger RNA-baseddelivery methods, or RNP-based delivery (ribonucleoprotein-baseddelivery). And, each of these methods of delivery may be more efficientand/or effective by dividing up the prime editor into smaller pieces.Once inside the cell, the smaller pieces can assemble into a functionalprime editor. Depending on the means of splitting, the divided primeeditor fragments can be reassembled in a non-covalent manner or acovalent manner to reform the prime editor. In one embodiment, the primeeditor can be split at one or more split sites into two or morefragments. The fragments can be unmodified (other than being split).Once the fragments are delivered to the cell (e.g., by direct deliveryof a ribonucleoprotein complex or by nucleic delivery—e.g., mRNAdelivery or virus vector based delivery), the fragments can reassociatecovalently or non-covalently to reconstitute the prime editor. Inanother embodiment, the prime editor can be split at one or more splitsites into two or more fragments. Each of the fragments can be modifiedto comprise a dimerization domain, whereby each fragment that is formedis coupled to a dimerization domain. Once delivered or expressed withina cell, the dimerization domains of the different fragments associateand bind to one another, bringing the different prime editor fragmentstogether to reform a functional prime editor. In yet another embodiment,the prime editor fragment may be modified to comprise a split intein.Once delivered or expressed within a cell, the split intein domains ofthe different fragments associate and bind to one another, and thenundergo trans-splicing, which results in the excision of thesplit-intein domains from each of the fragments, and a concomitantformation of a peptide bond between the fragments, thereby restoring theprime editor.

In one embodiment, the prime editor can be delivered using asplit-intein approach.

The location of the split site can be positioned between any one or morepair of residues in the prime editor and in any domains therein,including within the napDNAbp domain, the polymerase domain (e.g., RTdomain), linker domain that joins the napDNAbp domain and the polymerasedomain.

In one embodiment, depicted in FIG. 66 , the prime editor (PE) isdivided at a split site within the napDNAbp.

In certain embodiments, the napDNAbp is a canonical SpCas9 polypeptideof SEQ ID NO: 18, as follows:

SpCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN SEQ ID NO: StreptococcusTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR 18 pyogenesKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH M1ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL SwissProtRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ AccessionTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL No.PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS Q99ZW2KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI Wild typeLRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 1368 AAPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

In certain embodiments, the SpCas9 is split into two fragments at asplit site located between residues 1 and 2, or 2 and 3, or 3 and 4, or4 and 5, or 5 and 6, or 6 and 7, or 7 and 8, or 8 and 9, or 9 and 10, orbetween any two pair of residues located anywhere between residues 1-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200,200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,1000-1100, 1100-1200, 1200-1300, or 1300-1368 of canonical SpCas9 of SEQID NO: 18.

In certain embodiments, a napDNAbp is split into two fragments at asplit site that is located at a pair of residue that corresponds to anytwo pair of residues located anywhere between positions 1-10, 10-20,20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200,200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,1000-1100, 1100-1200, 1200-1300, or 1300-1368 of canonical SpCas9 of SEQID NO: 18.

In certain embodiments, the SpCas9 is split into two fragments at asplit site located between residues 1 and 2, or 2 and 3, or 3 and 4, or4 and 5, or 5 and 6, or 6 and 7, or 7 and 8, or 8 and 9, or 9 and 10, orbetween any two pair of residues located anywhere between residues 1-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200,200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,1000-1100, 1100-1200, 1200-1300, or 1300-1368 of canonical SpCas9 of SEQID NO: 18. In certain embodiments, the split site is located one or morepolypeptide bond sites (i.e., a “split site or split-intein splitsite”), fused to a split intein, and then delivered to cells asseparately-encoded fusion proteins. Once the split-intein fusionproteins (i.e., protein halves) are expressed within a cell, theproteins undergo trans-splicing to form a complete or whole PE with theconcomitant removal of the joined split-intein sequences.

For example, as shown in FIG. 66 , the N-terminal extein can be fused toa first split-intein (e.g., N intein) and the C-terminal extein can befused to a second split-intein (e.g., C intein). The N-terminal exteinbecomes fused to the C-terminal extein to reform a whole prime editorfusion protein comprising an napDNAbp domain and a polymerase domain(e.g., RT domain) upon the self-association of the N intein and the Cintein inside the cell, followed by their self-excision, and theconcomitant formation of a peptide bond between the N-terminal exteinand C-terminal extein portions of a whole prime editor (PE).

To take advantage of a split-PE delivery strategy using split-inteins,the prime editor needs to be divided at one or more split sites tocreate at least two separate halves of a prime editor, each of which maybe rejoined inside a cell if each half is fused to a split-inteinsequence.

In certain embodiments, the prime editor is split at a single splitsite. In certain other embodiments, the prime editor is split at twosplit sites, or three split sites, or four split sites, or more.

In a preferred embodiment, the prime editor is split at a single splitsite to create two separate halves of a prime editor, each of which canbe fused to a split intein sequence

An exemplary split intein is the Ssp DnaE intein, which comprises twosubunits, namely, DnaE-N and DnaE-C. The two different subunits areencoded by separate genes, namely dnaE-n and dnaE-c, which encode theDnaE-N and DnaE-C subunits, respectively. DnaE is a naturally occurringsplit intein in Synechocystis sp. PCC6803 and is capable of directingtrans-splicing of two separate proteins, each comprising a fusion witheither DnaE-N or DnaE-C.

Additional naturally occurring or engineered split-intein sequences areknown in the or can be made from whole-intein sequences described hereinor those available in the art. Examples of split-intein sequences can befound in Stevens et al., “A promiscuous split intein with expandedprotein engineering applications,” PNAS, 2017, Vol. 114: 8538-8543; Iwaiet al., “Highly efficient protein trans-splicing by a naturally splitDnaE intein from Nostoc punctiforme, FEBS Lett, 580: 1853-1858, each ofwhich are incorporated herein by reference. Additional split inteinsequences can be found, for example, in WO 2013/045632, WO 2014/055782,WO 2016/069774, and EP2877490, the contents each of which areincorporated herein by reference.

In addition, protein splicing in trans has been described in vivo and invitro (Shingledecker, et al., Gene 207:187 (1998), Southworth, et al.,EMBO J. 17:918 (1998); Mills, et al., Proc. Natl. Acad. Sci. USA,95:3543-3548 (1998); Lew, et al., J. Biol. Chem., 273:15887-15890(1998); Wu, et al., Biochim. Biophys. Acta 35732:1 (1998b), Yamazaki, etal., J. Am. Chem. Soc. 120:5591 (1998), Evans, et al., J. Biol. Chem.275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999);Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc.Natl. Acad. Sci. USA 96:13638-13643 (1999)) and provides the opportunityto express a protein as to two inactive fragments that subsequentlyundergo ligation to form a functional product, e.g., as shown in FIGS.66 and 67 with regard to the formation of a complete PE fusion proteinfrom two separately-expressed halves.

In various embodiments described herein, the continuous evolutionmethods (e.g., PACE) may be used to evolve a first portion of a baseeditor. A first portion could include a single component or domain,e.g., a Cas9 domain, a deaminase domain, or a UGI domain. The separatelyevolved component or domain can be then fused to the remaining portionsof the base editor within a cell by separately express both the evolvedportion and the remaining non-evolved portions with split-inteinpolypeptide domains. The first portion could more broadly include anyfirst amino acid portion of a base editor that is desired to be evolvedusing a continuous evolution method described herein. The second portionwould in this embodiment refer to the remaining amino acid portion ofthe base editor that is not evolved using the herein methods. Theevolved first portion and the second portion of the base editor couldeach be expressed with split-intein polypeptide domains in a cell. Thenatural protein splicing mechanisms of the cell would reassemble theevolved first portion and the non-evolved second portion to form asingle fusion protein evolved base editor. The evolved first portion maycomprise either the N- or C-terminal part of the single fusion protein.In an analogous manner, use of a second orthogonal trans-splicing inteinpair could allow the evolved first portion to comprise an internal partof the single fusion protein.

Thus, any of the evolved and non-evolved components of the base editorsherein described may be expressed with split-intein tags in order tofacilitate the formation of a complete base editor comprising theevolved and non-evolved component within a cell.

The mechanism of the protein splicing process has been studied in greatdetail (Chong, et al., J. Biol. Chem. 1996, 271, 22159-22168; Xu, M-Q &Perler, F. B. EMBO Journal, 1996, 15, 5146-5153) and conserved aminoacids have been found at the intein and extein splicing points (Xu, etal., EMBO Journal, 1994, 13 5517-522). The constructs described hereincontain an intein sequence fused to the 5′-terminus of the first gene(e.g., the evolved portion of the base editor). Suitable inteinsequences can be selected from any of the proteins known to containprotein splicing elements. A database containing all known inteins canbe found on the World Wide Web (Perler, F. B. Nucleic Acids Research,1999, 27, 346-347). The intein sequence is fused at the 3′ end to the 5′end of a second gene. For targeting of this gene to a certain organelle,a peptide signal can be fused to the coding sequence of the gene. Afterthe second gene, the intein-gene sequence can be repeated as often asdesired for expression of multiple proteins in the same cell. Formulti-intein containing constructs, it may be useful to use inteinelements from different sources. After the sequence of the last gene tobe expressed, a transcription termination sequence must be inserted. Inone embodiment, a modified intein splicing unit is designed so that itcan both catalyze excision of the exteins from the inteins as well asprevent ligation of the exteins. Mutagenesis of the C-terminal exteinjunction in the Pyrococcus species GB-D DNA polymerase was found toproduce an altered splicing element that induces cleavage of exteins andinteins but prevents subsequent ligation of the exteins (Xu, M-Q &Perler, F. B. EMBO Journal, 1996, 15, 5146-5153). Mutation of serine 538to either an alanine or glycine induced cleavage but prevented ligation.Mutation of equivalent residues in other intein splicing units shouldalso prevent extein ligation due to the conservation of amino acids atthe C-terminal extein junction to the intein. A preferred intein notcontaining an endonuclease domain is the Mycobacterium xenopi GyrAprotein (Telenti, et al. J. Bacteriol. 1997, 179, 6378-6382). Othershave been found in nature or have been created artificially by removingthe endonuclease domains from endonuclease containing inteins (Chong, etal. J. Biol. Chem. 1997, 272, 15587-15590). In a preferred embodiment,the intein is selected so that it consists of the minimal number ofamino acids needed to perform the splicing function, such as the inteinfrom the Mycobacterium xenopi GyrA protein (Telenti, A., et al., J.Bacteriol. 1997, 179, 6378-6382). In an alternative embodiment, anintein without endonuclease activity is selected, such as the inteinfrom the Mycobacterium xenopi GyrA protein or the Saccharomycescerevisiae VMA intein that has been modified to remove endonucleasedomains (Chong, 1997). Further modification of the intein splicing unitmay allow the reaction rate of the cleavage reaction to be alteredallowing protein dosage to be controlled by simply modifying the genesequence of the splicing unit.

Inteins can also exist as two fragments encoded by two separatelytranscribed and translated genes. These so-called split inteinsself-associate and catalyze protein-splicing activity in trans. Splitinteins have been identified in diverse cyanobacteria and archaea (Caspiet al, Mol Microbiol. 50: 1569-1577 (2003); Choi J. et al, J Mol Biol.556: 1093-1106 (2006.); Dassa B. et al, Biochemistry. 46:322-330(2007.); Liu X. and Yang J., J Biol Chem. 275:26315-26318 (2003); Wu H.et al.

Proc Natl Acad Sci USA. £5:9226-9231 (1998.); and Zettler J. et al, FEBSLetters. 553:909-914 (2009)), but have not been found in eukaryotes thusfar. Recently, a bioinformatic analysis of environmental metagenomicdata revealed 26 different loci with a novel genomic arrangement. Ateach locus, a conserved enzyme coding region is interrupted by a splitintein, with a freestanding endonuclease gene inserted between thesections coding for intein subdomains. Among them, five loci werecompletely assembled: DNA helicases (gp41-1, gp41-8);Inosine-5′-monophosphate dehydrogenase (IMPDH-1); and Ribonucleotidereductase catalytic subunits (NrdA-2 and NrdJ-1). This fractured geneorganization appears to be present mainly in phages (Dassa et al,Nucleic Acids Research. 57:2560-2573 (2009)).

The split intein Npu DnaE was characterized as having the highest ratereported for the protein trans-splicing reaction. In addition, the NpuDnaE protein splicing reaction is considered robust and high-yieldingwith respect to different extein sequences, temperatures from 6 to 37°C., and the presence of up to 6M Urea (Zettler J. et al, FEBS Letters.553:909-914 (2009); Iwai I. et al, FEBS Letters 550: 1853-1858 (2006)).As expected, when the Cysl Ala mutation at the N-domain of these inteinswas introduced, the initial N to S-acyl shift and therefore proteinsplicing was blocked. Unfortunately, the C-terminal cleavage reactionwas also almost completely inhibited. The dependence of the asparaginecyclization at the C-terminal splice junction on the acyl shift at theN-terminal scissile peptide bond seems to be a unique property common tothe naturally split DnaE intein alleles (Zettler J. et al. FEBS Letters.555:909-914 (2009)).

The mechanism of protein splicing typically has four steps [29-30]: 1)an N—S or N—O acyl shift at the intein N-terminus, which breaks theupstream peptide bond and forms an ester bond between the N-extein andthe side chain of the intein's first amino acid (Cys or Ser); 2) atransesterification relocating the N-extein to the intein C-terminus,forming a new ester bond linking the N-extein to the side chain of theC-extein's first amino acid (Cys, Ser, or Thr); 3) Asn cyclizationbreaking the peptide bond between the intein and the C-extein; and 4) aS-N or O-N acyl shift that replaces the ester bond with a peptide bondbetween the N-extein and C-extein.

Protein trans-splicing, catalyzed by split inteins, provides an entirelyenzymatic method for protein ligation [31]. A split-intein isessentially a contiguous intein (e.g. a mini-intein) split into twopieces named N-intein and C-intein, respectively. The N-intein andC-intein of a split intein can associate non-covalently to form anactive intein and catalyze the splicing reaction essentially in same wayas a contiguous intein does. Split inteins have been found in nature andalso engineered in laboratories [31-35]. As used herein, the term “splitintein” refers to any intein in which one or more peptide bond breaksexists between the N-terminal and C-terminal amino acid sequences suchthat the N-terminal and C-terminal sequences become separate moleculesthat can non-covalently reassociate, or reconstitute, into an inteinthat is functional for trans-splicing reactions. Any catalyticallyactive intein, or fragment thereof, may be used to derive a split inteinfor use in the methods of the invention. For example, in one aspect thesplit intein may be derived from a eukaryotic intein. In another aspect,the split intein may be derived from a bacterial intein. In anotheraspect, the split intein may be derived from an archaeal intein.Preferably, the split intein so-derived will possess only the amino acidsequences essential for catalyzing trans-splicing reactions.

As used herein, the “N-terminal split intein (In)” refers to any inteinsequence that comprises an N-terminal amino acid sequence that isfunctional for trans-splicing reactions. An In thus also comprises asequence that is spliced out when trans-splicing occurs. An In cancomprise a sequence that is a modification of the N-terminal portion ofa naturally occurring intein sequence. For example, an In can compriseadditional amino acid residues and/or mutated residues so long as theinclusion of such additional and/or mutated residues does not render theIn non-functional in trans-splicing. Preferably, the inclusion of theadditional and/or mutated residues improves or enhances thetrans-splicing activity of the In.

As used herein, the “C-terminal split intein (Ic)” refers to any inteinsequence that comprises a C-terminal amino acid sequence that isfunctional for trans-splicing reactions. In one aspect, the Ic comprises4 to 7 contiguous amino acid residues, at least 4 amino acids of whichare from the last β-strand of the intein from which it was derived. AnIc thus also comprises a sequence that is spliced out whentrans-splicing occurs. An Ic can comprise a sequence that is amodification of the C-terminal portion of a naturally occurring inteinsequence. For example, an Ic can comprise additional amino acid residuesand/or mutated residues so long as the inclusion of such additionaland/or mutated residues does not render the In non-functional intrans-splicing. Preferably, the inclusion of the additional and/ormutated residues improves or enhances the trans-splicing activity of theIc.

In some embodiments of the invention, a peptide linked to an Ic or an Incan comprise an additional chemical moiety including, among others,fluorescence groups, biotin, polyethylene glycol (PEG), amino acidanalogs, unnatural amino acids, phosphate groups, glycosyl groups,radioisotope labels, and pharmaceutical molecules. In other embodiments,a peptide linked to an Ic can comprise one or more chemically reactivegroups including, among others, ketone, aldehyde, Cys residues and Lysresidues. The N-intein and C-intein of a split intein can associatenon-covalently to form an active intein and catalyze the splicingreaction when an “intein-splicing polypeptide (ISP)” is present. As usedherein, “intein-splicing polypeptide (ISP)” refers to the portion of theamino acid sequence of a split intein that remains when the Ic, In, orboth, are removed from the split intein. In certain embodiments, the Incomprises the ISP. In another embodiment, the Ic comprises the ISP. Inyet another embodiment, the ISP is a separate peptide that is notcovalently linked to In nor to Ic.

Split inteins may be created from contiguous inteins by engineering oneor more split sites in the unstructured loop or intervening amino acidsequence between the −12 conserved beta-strands found in the structureof mini-inteins [25-28]. Some flexibility in the position of the splitsite within regions between the beta-strands may exist, provided thatcreation of the split will not disrupt the structure of the intein, thestructured beta-strands in particular, to a sufficient degree thatprotein splicing activity is lost.

In protein trans-splicing, one precursor protein consists of an N-exteinpart followed by the N-intein, another precursor protein consists of theC-intein followed by a C-extein part, and a trans-splicing reaction(catalyzed by the N- and C-inteins together) excises the two inteinsequences and links the two extein sequences with a peptide bond.Protein trans-splicing, being an enzymatic reaction, can work with verylow (e.g. micromolar) concentrations of proteins and can be carried outunder physiological conditions.

[2] Other Programmable Nucleases

In various embodiments described herein, the prime editors comprise anapDNAbp, such as a Cas9 protein. These proteins are “programmable” byway of their becoming complexed with a guide RNA (or a PEgRNA, as thecase may be), which guides the Cas9 protein to a target site on the DNAwhich possess a sequence that is complementary to the spacer portion ofthe gRNA (or PEgRNA) and also which possesses the required PAM sequence.However, in certain embodiment envisioned here, the napDNAbp may besubstituted with a different type of programmable protein, such as azinc finger nuclease or a transcription activator-like effector nuclease(TALEN).

FIG. 1J depicts such a variation of prime editing contemplated hereinthat replaces the napDNAbp (e.g., SpCas9 nickase) with any programmablenuclease domain, such as zinc finger nucleases (ZFN) or transcriptionactivator-like effector nucleases (TALEN). As such, it is contemplatedthat suitable nucleases do not necessarily need to be “programmed” by anucleic acid targeting molecule (such as a guide RNA), but rather, maybe programmed by defining the specificity of a DNA-binding domain, suchas and in particular, a nuclease. Just as in prime editing with napDNAbpmoities, it is preferable that such alternative programmable nucleasesbe modified such that only one strand of a target DNA is cut. In otherwords, the programmable nucleases should function as nickases,preferably. Once a programmable nuclease is selected (e.g., a ZFN or aTALEN), then additional functionalities may be engineered into thesystem to allow it to operate in accordance with a prime editing-likemechanism. For example, the programmable nucleases may be modified bycoupling (e.g., via a chemical linker) an RNA or DNA extension armthereto, wherein the extension arm comprises a primer binding site (PBS)and a DNA synthesis template. The programmable nuclease may also becoupled (e.g., via a chemical or amino acid linker) to a polymerase, thenature of which will depend upon whether the extension arm is DNA orRNA. In the case of an RNA extension arm, the polymerase can be anRNA-dependent DNA polymerase (e.g., reverse transcriptase). In the caseof a DNA extension arm, the polymerase can be a DNA-dependent DNApolymerase (e.g., a prokaryotic polymerase, including Pol I, Pol II, orPol III, or a eukaryotic polymerase, including Pol a, Pol b, Pol g, Pold, Pol e, or Pol z). The system may also include other functionalitiesadded as fusions to the programmable nucleases, or added in trans tofacilitate the reaction as a whole (e.g., (a) a helicase to unwind theDNA at the cut site to make the cut strand with the 3′ end available asa primer, (b) a FEN1 to help remove the endogenous strand on the cutstrand to drive the reaction towards replacement of the endogenousstrand with the synthesized strand, or (c) a nCas9:gRNA complex tocreate a second site nick on the opposite strand, which may help drivethe integration of the synthesize repair through favored cellular repairof the non-edited strand). In an analogous manner to prime editing witha napDNAbp, such a complex with an otherwise programmable nuclease couldbe used to synthesize and then install a newly synthesized replacementstrand of DNA carrying an edit of interest permanently into a targetsite of DNA.

Suitable alternative programmable nucleases are well known in the artwhich may be used in place of a napDNAbp:gRNA complex to construct analternative prime editor system that can be programmed to selectivelybind a target site of DNA, and which can be further modified in themanner described above to co-localize a polymerase and an RNA or DNAextension arm comprising a primer binding site and a DNA synthesistemplate to specific nick site. For example, and as represented in FIG.1J, Transcription Activator-Like Effector Nucleases (TALENs) may be usedas the programmable nuclease in the prime editing methods andcompositions of matter described herein. TALENS are artificialrestriction enzymes generated by fusing the TAL effector DNA bindingdomain to a DNA cleavage domain. These reagents enable efficient,programmable, and specific DNA cleavage and represent powerful tools forgenome editing in situ. Transcription activator-like effectors (TALEs)can be quickly engineered to bind practically any DNA sequence. The termTALEN, as used herein, is broad and includes a monomeric TALEN that cancleave double stranded DNA without assistance from another TALEN. Theterm TALEN is also used to refer to one or both members of a pair ofTALENs that are engineered to work together to cleave DNA at the samesite. TALENs that work together may be referred to as a left-TALEN and aright-TALEN, which references the handedness of DNA. See U.S. Ser. No.12/965,590; U.S. Ser. No. 13/426,991 (U.S. Pat. No. 8,450,471); U.S.Ser. No. 13/427,040 (U.S. Pat. No. 8,440,431); U.S. Ser. No. 13/427,137(U.S. Pat. No. 8,440,432); and U.S. Ser. No. 13/738,381, all of whichare incorporated by reference herein in their entirety. In addition,TALENS are described in WO 2015/027134, U.S. Pat. No. 9,181,535, Boch etal., “Breaking the Code of DNA Binding Specificity of TAL-Type IIIEffectors”, Science, vol. 326, pp. 1509-1512 (2009), Bogdanove et al.,TAL Effectors: Customizable Proteins for DNA Targeting, Science, vol.333, pp. 1843-1846 (2011), Cade et al., “Highly efficient generation ofheritable zebrafish gene mutations using homo- and heterodimericTALENs”, Nucleic Acids Research, vol. 40, pp. 8001-8010 (2012), andCermak et al., “Efficient design and assembly of custom TALEN and otherTAL effector-based constructs for DNA targeting”, Nucleic AcidsResearch, vol. 39, No. 17, e82 (2011), each of which are incorporatedherein by reference.

As represented in FIG. 1J, zinc finger nucleases may also be used asalternative programmable nucleases for use in prime editing in place ofnapDNAbps, such as Cas9 nickases. Like with TALENS, the ZFN proteins maybe modified such that they function as nickases, i.e., engineering theZFN such that it cleaves only one strand of the target DNA in a mannersimilar to the napDNAbp used with the prime editors described herein.ZFN proteins have been extensively described in the art, for example, inCarroll et al., “Genome Engineering with Zinc-Finger Nucleases,”Genetics, August 2011, Vol. 188: 773-782; Durai et al., “Zinc fingernucleases: custom-designed molecular scissors for genome engineering ofplant and mammalian cells,” Nucleic Acids Res, 2005, Vol. 33: 5978-90;and Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based methods for genomeengineering,” Trends Biotechnol. 2013, Vol. 31: 397-405, each of whichare incorporated herein by reference in their entireties.

[3] Polymerases (e.g., Reverse Transcriptases)

In various embodiments, the prime editor (PE) system disclosed hereinincludes a polymerase (e.g., DNA-dependent DNA polymerase orRNA-dependent DNA polymerase, such as, reverse transcriptase), or avariant thereof, which can be provided as a fusion protein with anapDNAbp or other programmable nuclease, or provide in trans.

Any polymerase may be used in the prime editors disclosed herein. Thepolymerases may be wild type polymerases, functional fragments, mutants,variants, or truncated variants, and the like. The polymerases mayinclude wild type polymerases from eukaryotic, prokaryotic, archaeal, orviral organisms, and/or the polymerases may be modified by geneticengineering, mutagenesis, directed evolution-based processes. Thepolymerases may include T7 DNA polymerase, T5 DNA polymerase, T4 DNApolymerase, Klenow fragment DNA polymerase, DNA polymerase III and thelike. The polymerases may also be thermostable, and may include Taq,Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNApolymerases, KOD, Tgo, JDF3, and mutants, variants and derivativesthereof (see U.S. Pat. Nos. 5,436,149; 4,889,818; 4,965,185; 5,079,352;5,614,365; 5,374,553; 5,270,179; 5,047,342; 5,512,462; WO 92/06188; WO92/06200; WO 96/10640; Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F.C., et al., PCR Meth. Appl. 2:275-287 (1993); Flaman, J.-M, et al., Nuc.Acids Res. 22(15):3259-3260 (1994), each of which are incorporated byreference). For synthesis of longer nucleic acid molecules (e.g, nucleicacid molecules longer than about 3-5 Kb in length), at least two DNApolymerases can be employed. In certain embodiments, one of thepolymerases can be substantially lacking a 3′ exonuclease activity andthe other may have a 3′ exonuclease activity. Such pairings may includepolymerases that are the same or different. Examples of DNA polymerasessubstantially lacking in 3′ exonuclease activity include, but are notlimited to, Taq, Tne(exo-), Tma(exo-), Pfu(exo-), Pwo(exo-), exo-KOD andTth DNA polymerases, and mutants, variants and derivatives thereof.

Preferably, the polymerase usable in the prime editors disclosed hereinare “template-dependent” polymerase (since the polymerases are intendedto rely on the DNA synthesis template to specify the sequence of the DNAstrand under synthesis during prime editing. As used herein, the term“template DNA molecule” refers to that strand of a nucleic acid fromwhich a complementary nucleic acid strand is synthesized by a DNApolymerase, for example, in a primer extension reaction of the DNAsynthesis template of a PEgRNA.

As used herein, the term “template dependent manner” is intended torefer to a process that involves the template dependent extension of aprimer molecule (e.g., DNA synthesis by DNA polymerase). The term“template dependent manner” refers to polynucleotide synthesis of RNA orDNA wherein the sequence of the newly synthesized strand ofpolynucleotide is dictated by the well-known rules of complementary basepairing (see, for example, Watson, J. D. et al., In: Molecular Biologyof the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987)).The term “complementary” refers to the broad concept of sequencecomplementarity between regions of two polynucleotide strands or betweentwo nucleotides through base-pairing. It is known that an adeninenucleotide is capable of forming specific hydrogen bonds (“basepairing”) with a nucleotide which is thymine or uracil. Similarly, it isknown that a cytosine nucleotide is capable of base pairing with aguanine nucleotide. As such, in the case of prime editing, it can besaid that the single strand of DNA synthesized by the polymerase of theprime editor against the DNA synthesis template is said to be“complementary” to the sequence of the DNA synthesis template.

A. Exemplary Polymerases

In various embodiments, the prime editors described herein comprise apolymerase. The disclosure contemplates any wild type polymeraseobtained from any naturally-occurring organism or virus, or obtainedfrom a commercial or non-commercial source. In addition, the polymerasesusable in the prime editors of the disclosure can include anynaturally-occurring mutant polymerase, engineered mutant polymerase, orother variant polymerase, including truncated variants that retainfunction. The polymerases usable herein may also be engineered tocontain specific amino acid substitutions, such as those specificallydisclosed herein. In certain preferred embodiments, the polymerasesusable in the prime editors of the disclosure are template-basedpolymerases, i.e., they synthesize nucleotide sequences in atemplate-dependent manner.

A polymerase is an enzyme that synthesizes a nucleotide strand and whichmay be used in connection with the prime editor systems describedherein. The polymerases are preferrably “template-dependent” polymerases(i.e., a polymerase which synthesizes a nucleotide strand based on theorder of nucleotide bases of a template strand). In certainconfigurations, the polymerases can also be a “template-independent”(i.e., a polymerase which synthesizes a nucleotide strand without therequirement of a template strand). A polymerase may also be furthercategorized as a “DNA polymerase” or an “RNA polymerase.” In variousembodiments, the prime editor system comprises a DNA polymerase. Invarious embodiments, the DNA polymerase can be a “DNA-dependent DNApolymerase” (i.e., whereby the template molecule is a strand of DNA). Insuch cases, the DNA template molecule can be a PEgRNA, wherein theextension arm comprises a strand of DNA. In such cases, the PEgRNA maybe referred to as a chimeric or hybrid PEgRNA which comprises an RNAportion (i.e., the guide RNA components, including the spacer and thegRNA core) and a DNA portion (i.e., the extension arm). In various otherembodiments, the DNA polymerase can be an “RNA-dependent DNA polymerase”(i.e., whereby the template molecule is a strand of RNA). In such cases,the PEgRNA is RNA, i.e., including an RNA extension. The term“polymerase” may also refer to an enzyme that catalyzes thepolymerization of nucleotide (i.e., the polymerase activity). Generally,the enzyme will initiate synthesis at the 3′-end of a primer annealed toa polynucleotide template sequence (e.g., such as a primer sequenceannealed to the primer binding site of a PEgRNA), and will proceedtoward the 5′ end of the template strand. A “DNA polymerase” catalyzesthe polymerization of deoxynucleotides. As used herein in reference to aDNA polymerase, the term DNA polymerase includes a “functional fragmentthereof”. A “functional fragment thereof” refers to any portion of awild-type or mutant DNA polymerase that encompasses less than the entireamino acid sequence of the polymerase and which retains the ability,under at least one set of conditions, to catalyze the polymerization ofa polynucleotide. Such a functional fragment may exist as a separateentity, or it may be a constituent of a larger polypeptide, such as afusion protein.

In some embodiments, the polymerases can be from bacteriophage.Bacteriophage DNA polymerases are generally devoid of 5′ to 3′exonuclease activity, as this activity is encoded by a separatepolypeptide. Examples of suitable DNA polymerases are T4, T7, and phi29DNA polymerase. The enzymes available commercially are: T4 (availablefrom many sources e.g., Epicentre) and T7 (available from many sources,e.g. Epicentre for unmodified and USB for 3′ to 5′ exo T7 “Sequenase”DNA polymerase).

The other embodiments, the polymerases are archaeal polymerases. Thereare 2 different classes of DNA polymerases which have been identified inarchaea: 1. Family B/pol I type (homologs of Pfu from Pyrococcusfuriosus) and 2. pol II type (homologs of P. furiosus DP1/DP2 2-subunitpolymerase). DNA polymerases from both classes have been shown tonaturally lack an associated 5′ to 3′ exonuclease activity and topossess 3′ to 5′ exonuclease (proofreading) activity. Suitable DNApolymerases (pol I or pol II) can be derived from archaea with optimalgrowth temperatures that are similar to the desired assay temperatures.

Thermostable archaeal DNA polymerases are isolated from Pyrococcusspecies (furiosus, species GB-D, woesii, abysii, horikoshii),Thermococcus species (kodakaraensis KOD1, litoralis, species 9 degreesNorth-7, species JDF-3, gorgonarius), Pyrodictium occultum, andArchaeoglobus fulgidus.

Polymerases may also be from eubacterial species. There are 3 classes ofeubacterial DNA polymerases, pol I, II, and III. Enzymes in the Pol IDNA polymerase family possess 5′ to 3′ exonuclease activity, and certainmembers also exhibit 3′ to 5′ exonuclease activity. Pol II DNApolymerases naturally lack 5′ to 3′ exonuclease activity, but do exhibit3′ to 5′ exonuclease activity. Pol III DNA polymerases represent themajor replicative DNA polymerase of the cell and are composed ofmultiple subunits. The pol III catalytic subunit lacks 5′ to 3′exonuclease activity, but in some cases 3′ to 5′ exonuclease activity islocated in the same polypeptide.

There are a variety of commercially available Pol I DNA polymerases,some of which have been modified to reduce or abolish 5′ to 3′exonuclease activity.

Suitable thermostable pol I DNA polymerases can be isolated from avariety of thermophilic eubacteria, including Thermus species andThermotoga maritima such as Thermus aquaticus (Taq), Thermusthermophilus (Tth) and Thermotoga maritima (Tma UlTma).

Additional eubacteria related to those listed above are described inThermophilic Bacteria (Kristjansson, J. K., ed.) CRC Press, Inc., BocaRaton, Fla., 1992.

The invention further provides for chimeric or non-chimeric DNApolymerases that are chemically modified according to methods disclosedin U.S. Pat. Nos. 5,677,152, 6,479,264 and 6,183,998, the contents ofwhich are hereby incorporated by reference in their entirety.

Additional archaea DNA polymerases related to those listed above aredescribed in the following references: Archaea: A Laboratory Manual(Robb, F. T. and Place, A. R., eds.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1995 and Thermophilic Bacteria(Kristjansson, J. K., ed.) CRC Press, Inc., Boca Raton, Fla., 1992.

B. Exemplary Reverse Transcriptases

In various embodiments, the prime editors described herein comprise areverse transcriptase as the polymerase. The disclosure contemplates anywild type reverse transcriptase obtained from any naturally-occurringorganism or virus, or obtained from a commercial or non-commercialsource. In addition, the reverse transcriptases usable in the primeeditors of the disclosure can include any naturally-occurring mutant RT,engineered mutant RT, or other variant RT, including truncated variantsthat retain function. The RTs may also be engineered to contain specificamino acid substitutions, such as those specifically disclosed herein.

Reverse transcriptases are multi-functional enzymes typically with threeenzymatic activities including RNA- and DNA-dependent DNA polymerizationactivity, and an RNaseH activity that catalyzes the cleavage of RNA inRNA-DNA hybrids. Some mutants of reverse transcriptases have disabledthe RNaseH moiety to prevent unintended damage to the mRNA. Theseenzymes that synthesize complementary DNA (cDNA) using mRNA as atemplate were first identified in RNA viruses. Subsequently, reversetranscriptases were isolated and purified directly from virus particles,cells or tissues. (e.g., see Kacian et al., 1971, Biochim. Biophys. Acta46: 365-83; Yang et al., 1972, Biochem. Biophys. Res. Comm. 47: 505-11;Gerard et al., 1975, J. Virol. 15: 785-97; Liu et al., 1977, Arch.Virol. 55 187-200; Kato et al., 1984, J. Virol. Methods 9: 325-39; Lukeet al., 1990, Biochem. 29: 1764-69 and Le Grice et al., 1991, J. Virol.65: 7004-07, each of which are incorporated by reference). Morerecently, mutants and fusion proteins have been created in the quest forimproved properties such as thermostability, fidelity and activity. Anyof the wild type, variant, and/or mutant forms of reverse transcriptasewhich are known in the art or which can be made using methods known inthe art are contemplated herein.

The reverse transcriptase (RT) gene (or the genetic informationcontained therein) can be obtained from a number of different sources.For instance, the gene may be obtained from eukaryotic cells which areinfected with retrovirus, or from a number of plasmids which containeither a portion of or the entire retrovirus genome. In addition,messenger RNA-like RNA which contains the RT gene can be obtained fromretroviruses. Examples of sources for RT include, but are not limitedto, Moloney murine leukemia virus (M-MLV or MLVRT); human T-cellleukemia virus type 1 (HTLV-1); bovine leukemia virus (BLV); RousSarcoma Virus (RSV); human immunodeficiency virus (HIV); yeast,including Saccharomyces, Neurospora, Drosophila; primates; and rodents.See, for example, Weiss, et al., U.S. Pat. No. 4,663,290 (1987); Gerard,G. R., DNA:271-79 (1986); Kotewicz, M. L., et al., Gene 35:249-58(1985); Tanese, N., et al., Proc. Natl. Acad. Sci. (USA):4944-48 (1985);Roth, M. J., at al., J. Biol. Chem. 260:9326-35 (1985); Michel, F., etal., Nature 316:641-43 (1985); Akins, R. A., et al., Cell 47:505-16(1986), EMBO J. 4:1267-75 (1985); and Fawcett, D. F., Cell 47:1007-15(1986) (each of which are incorporated herein by reference in theirentireties).

Wild Type RTs

Exemplary enzymes for use with the herein disclosed prime editors caninclude, but are not limited to, M-MLV reverse transcriptase and RSVreverse transcriptase. Enzymes having reverse transcriptase activity arecommercially available. In certain embodiments, the reversetranscriptase provided in trans to the other components of the primeeditor (PE) system. That is, the reverse transcriptase is expressed orotherwise provided as an individual component, i.e., not as a fusionprotein with a napDNAbp.

A person of ordinary skill in the art will recognize that wild typereverse transcriptases, including but not limited to, Moloney MurineLeukemia Virus (M-MLV); Human Immunodeficiency Virus (HIV) reversetranscriptase and avian Sarcoma-Leukosis Virus (ASLV) reversetranscriptase, which includes but is not limited to Rous Sarcoma Virus(RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reversetranscriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAVreverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper VirusMCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T)Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 HelperVirus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper VirusYAV reverse transcriptase, Rous Associated Virus (RAV) reversetranscriptase, and Myeloblastosis Associated Virus (MAV) reversetranscriptase may be suitably used in the subject methods andcomposition described herein.

Exemplary wild type RT enzymes are as follows:

DESCRIPTION SEQUENCE REVERSE TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLATRANSCRIPTASE VRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGIL(M-MLV RT) WILD VPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTV TYPEPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEW MOLONEY MURINERDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHP LEUKEMIA VIRUSDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAK USED IN PE1 (PRIMEKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPR EDITOR 1 FUSIONQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ PROTEINQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQ DISCLOSEDKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGK HEREIN)LTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP(SEQ ID NO: 89) REVERSEAFPLERPDWDYTTQAGRNHLVHYRQLLLAGLQNAGRSPTNL TRANSCRIPTASEAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETNVS MOLONEY MURINEMSFIWQSAPDIGRKLGRLEDLKSKTLGDLVREAEKIFNKRETP LEUKEMIA VIRUSEEREERIRRETEEKEERRRTVDEQKEKERDRRRHREMSKLLAT REF SEQ.VVIGQEQDRQEGERKRPQLDKDQCAYCKEKGHWAKDCPKKP AAA66622.1RGPRGPRPQTSLLTLGDXGGQGQDPPPEPRITLKVGGQPVTFLVDTGAQHSVLTQNPGPLSDKSAWVQGATGGKRYRWTTDRKVHLATGKVTHSFLHVPDCPYPLLGRDLLTKLKAQIHFEGSGAQVVGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGFTWLSDFPQAWAESGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEAL HRDLADFR(SEQ ID NO: 90)REVERSE TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGTAH TRANSCRIPTASECQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGIL FELINE LEUKEMIAKPCQSPWNTPLLPVKKPGTEDYRPVQDLREVNKRVEDIHPTV VIRUSPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEW REF SEQ. NP955579.1RDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNSRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFEDIKKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGGNHHDCLQILAETHGTRPDLTDQPLPDADLTWYTDGSSFIRNGEREAGAAVTTESEVIWAAPLPPGTSAQRAELIALTQALKMAEGKKLTVYTDSRYAFATTHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQ AKGNRLADDTAKKAATETHSSLTVLSEQ ID NO: 91) REVERSE PISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKETRANSCRIPTASE GKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFHIV-1 RT, CHAIN A WEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAREF SEQ. ITL3-A FTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGLTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLXKLLRGTKALTEVIPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKITTESIVIWGKTPKFKLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVDGAANRETKLGKAGYVTNRGRQKVVTLTDTTNQKTELQAIYLALQDSGLEVNIVTDSQYALGIIQAQPDQSESELVNQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSA GIRKV(SEQ ID NO: 92)SEE MARTINELLI ET AL., VIROLOGY, 1990, 174(1):135-144, WHICH IS INCORPORATED BY REFERENCE REVERSEPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKE TRANSCRIPTASEGKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDF HIV-1 RT, CHAIN BWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTA REF SEQ. ITL3-BFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGLTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGTKALTEVIPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKITTESIVIWGKTPKFKLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQ LEKEPIVGAETF(SEQ ID NO: 93)SEE STAMMERS ET AL., J. MOL. BIOL., 1994,242(4): 586-588, WHICH IS INCORPORATED BY REFERENCE REVERSETVALHLAIPLKWKPNHTPVWIDQWPLPEGKLVALTQLVEKEL TRANSCRIPTASEQLGHIEPSLSCWNTPVFVIRKASGSYRLLHDLRAVNAKLVPFG ROUS SARCOMAAVQQGAPVLSALPRGWPLMVLDLKDCFFSIPLAEQDREAFAF VIRUS RTTLPSVNNQAPARRFQWKVLPQGMTCSPTICQLIVGQILEPLRL REF SEQ. ACL14945KHPSLRMLHYMDDLLLAASSHDGLEAAGEEVISTLERAGFTISPDKVQKEPGVQYLGYKLGSTYAAPVGLVAEPRIATLWDVQKLVGSLQWLRPALGIPPRLRGPFYEQLRGSDPNEAREWNLDMKMAWREIVQLSTTAALERWDPALPLEGAVARCEQGAIGVLGQGLSTHPRPCLWLFSTQPTKAFTAWLEVLTLLITKLRASAVRTFGKEVDILLLPACFRDELPLPEGILLALRGFAGKIRSSDTPSIFDIARPLHVSLKVRVTDHPVPGPTVFTDASSSTHKGVVVWREGPRWEIKEIADLGASVQQLEARAVAMALLLWPTTPTNVVTDSAFVAKMLLKMGQEGVPSTAAAFILEDALSQRSAMAAVLHVRSHSEVPGFFTEGNDVADSQATFQAYPLREAKDLHTALHIGPRALSKACNISMQQAREVVQTCPHCNSAPALEAGVNPRGLGPLQIWQTDFTLEPRMAPRSWLAVTVDTASSAIVVTQHGRVTSVAAQHHWATVIAVLGRPKAIKTDNGSCFTSKSTREWLARWGIAHTTGIPGNSQGQAMVERANRLLKDKIRVLAEGDGFMKRIPTSKQGELLAKAMYALNHFERGENTKTPIQKHWRPTVLTEGPPVKIRIETGEWEKGWNVLVWGRGYAAVKNRDTDKVIWVPSRKVKPDIAQKDEVT KKDEASPLFA(SEQ ID NO: 94)SEE YASUKAWA ET AL., J. BIOCHEM. 2009, 145(3):315-324, WHICH IS INCORPORATED BY REFERENCE REVERSEMMDHLLQKTQIQNQTEQVMNITNPNSIYIKGRLYFKGYKKIEL TRANSCRIPTASEHCFVDTGASLCIASKFVIPEEHWINAERPIMVKIADGSSITINKV CAULIFLOWERCRDIDLIIAGEIFHIPTVYQQESGIDFIIGNNFCQLYEPFIQFTDRV MOSAIC VIRUS RTIFTKDRTYPVHIAKLTRAVRVGTEGFLESMKKRSKTQQPEPVNI REF SEQ. AGT42196STNKIAILSEGRRLSEEKLFITQQRMQKIEELLEKVCSENPLDPNKTKQWMKASIKLSDPSKAIKVKPMKYSPMDREEFDKQIKELLDLKVIKPSKSPHMAPAFLVNNEAEKRRGKKRMVVNYKAMNKATVGDAYNLPNKDELLTLIRGKKIFSSFDCKSGFWQVLLDQDSRPLTAFTCPQGHYEWNVVPFGLKQAPSIFQRHMDEAFRVFRKFCCVYVDDILVFSNNEEDHLLHVAMILQKCNQHGIILSKKKAQLFKKKINFLGLEIDEGTHKPQGHILEHINKFPDTLEDKKQLQRFLGILTYASDYIPKLAQIRKPLQAKLKENVPWKWTKEDTLYMQKVKKNLQGFPPLHHPLPEEKLIIETDASDDYWGGMLKAIKINEGTNTELICRYASGSFKAAEKNYHSNDKETLAVINTIKKFSIYLTPVHFLIRTDNTHFKSFVNLNYKGDSKLGRNIRWQAWLSHYSFDVEHIKGTDNHFADFLSREFNRVNS(SEQ ID NO: 95)SEE FARZADFAR ET AL., VIRUS GENES, 2013, 47(2):347-356, WHICH IS INCORPORATED BY REFERENCE REVERSEMKEKISKIDKNFYTDIFIKTSFQNEFEAGGVIPPIAKNQVSTISN TRANSCRIPTASEKNKTFYSLAHSSPHYSIQTRIEKFLLKNIPLSASSFAFRKERSYL KLEBSIELLAHYLEPHTQNVKYCHLDIVSFFHSIDVNIVRDTFSVYFSDEFLVK PNEUMONIAEKQSLLDAFMASVTLTAELDGVEKTFIPMGFKSSPSISNIIFRKI REF SEQ. RFF81513.1DILIQKFCDKNKITYTRYADDLLFSTKKENNILSSTFFINEISSILSINKFKLNKSKYLYKEGTISLGGYVIENILKDNSSGNIRLSSSKLNPLYKALYEIKKGSSSKHICIKVFNLKLKRFIYKKNKEKFEAKFYSSQLKNKLLGYRSYLLSFVIFHKKYKCINPIFLEKCVFLISEIE SIMNRKF(SEQ ID NO: 96)REVERSE MKITSNNVTAVINGKGWHSINWKKCHQHVKTIQTRIAKAACN TRANSCRIPTASEQQWRTVGRLQRLLVRSFSARALAVKRVTENSGRKTPGVDGQI ESCERICHIA COLIWSTPESKWEAIFKLRRKGYKPLPLKRVFIPKSNGKKRPLGIPV RTMLDRAMQALHLLGLEPVSETNADHNSYGFRPARCTADAIQQ REF SEQ. TGH57013 VCNMYSSRNASKWVLEGDIKGCFEHISHEWLLENIPMDKQILRNWLKAGIIEKSIFSKTLSGTPQGGIISPVLANMALDGLERLLQ NRFGRNRLI(SEQ ID NO: 97)REVERSE MSKIKINYEKYHIKPFPHFDQRIKVNKKVKENLQNPFYIAAHS TRANSCRIPTASEFYPFIHYKKISYKFKNGTLSSPKERDIFYSGHMDGYIYKHYGEI BACILLUS SUBTILISLNHKYNNTCIGKGIDHVSLAYRNNKMGKSNIHFAAEVINFISE RTQQQAFIFVSDFSSYFDSLDHAILKEKLIEVLEEQDKLSKDWWN REF SEQ. QB J66766VFKHITRYNWVEKEEVISDLECTKEKIARDKKSRERYYTPAEFREFRKRVNIKSNDTGVGIPQGTAISAVLANVYAIDLDQKLNQYALKYGGIYRRYSDDIIMVLPMTSDGQDPSNDHVSFIKSVVKRNKVTMGDSKTSVLYYANNNIYEDYQRKRESKMDYLGFSFDGMTVKIREKSLFKYYHRTYKKINSINWASVKKEKKVGRKKLYLLYSHLGRNYKGHGNFISYCKKAHAVFEGNKKIESLINQQIKRH WKKIQKRLVDV(SEQ ID NO: 98)EUBACTERIUM DTSNLMEQILSSDNLNRAYLQVVRNKGAEGVDGMKYTELKE RECTALE GROUP IIHLAKNGETIKGQLRTRKYKPQPARRVEIPKPDGGVRNLGVPT INTRON RTVTDRFIQQAIAQVLTPIYEEQFHDHSYGFRPNRCAQQAILTALNIMNDGNDWIVDIDLEKFFDTVNHDKLMTLIGRTIKDGDVISIVRKYLVSGIMIDDEYEDSIVGTPQGGNLSPLLANIMLNELDKEMEKRGLNFVRYADDCIIMVGSEMSANRVMRNISRFIEEKLGLKVNMTKSKVDRPSGLKYLGFGFYFDPRAHQFKAKPHAKSVAKFKKRMKELTCRSWGVSNSYKVEKLNQLIRGWINYFKIGSMKTLCKELDSRIRYRLRMCIWKQWKTPQNQEKNLVKLGIDRNTARRVAYTGKRIAYVCNKGAVNVAISNKRLASFGLISMLDYYIEKCV TC(SEQ ID NO: 99)GEOBACILLUS ALLERILARDNLITALKRVEANQGAPGIDGVSTDQLRDYIRAHSTEAROTHERMOPHILUS WSTIHAQLLAGTYRPAPVRRVEIPKPGGGTRQLGIPTVVDRLIQ GROUP IIQAILQELTPIFDPDFSSSSFGFRPGRNAHDAVRQAQGYIQEGYR INTRON RTYVVDMDLEKFFDRVNHDILMSRVARKVKDKRVLKLIRAYLQAGVMIEGVKVQTEEGTPQGGPLSPLLANILLDDLDKELEKRGLKFCRYADDCNIYVKSLRAGQRVKQSIQRFLEKTLKLKVNEEKSAVDRPWKRAFLGFSFTPERKARIRLAPRSIQRLKQRIRQLTNPNWSISMPERIHRVNQYVMGWIGYFRLVETPSVLQTIEGWIRRRLRLCQWLQWKRVRTRIRELRALGLKETAVMEIANTRKGAWRTTKTPQLHQALGKTYWTAQGLKSLTQR(SEQ ID NO: 100)

Variant and Error-Prone RTs

Reverse transcriptases are essential for synthesizing complementary DNA(cDNA) strands from RNA templates. Reverse transcriptases are enzymescomposed of distinct domains that exhibit different biochemicalactivities. The enzymes catalyze the synthesis of DNA from an RNAtemplate, as follows: In the presence of an annealed primer, reversetranscriptase binds to an RNA template and initiates the polymerizationreaction. RNA-dependent DNA polymerase activity synthesizes thecomplementary DNA (cDNA) strand, incorporating dNTPs. RNase H activitydegrades the RNA template of the DNA:RNA complex. Thus, reversetranscriptases comprise (a) a binding activity that recognizes and bindsto a RNA/DNA hybrid, (b) an RNA-dependent DNA polymerase activity, and(c) an RNase H activity. In addition, reverse transcriptases generallyare regarded as having various attributes, including theirthermostability, processivity (rate of dNTP incorporation), and fidelity(or error-rate). The reverse transcriptase variants contemplated hereinmay include any mutations to reverse transcriptase that impacts orchanges any one or more of these enzymatic activities (e.g.,RNA-dependent DNA polymerase activity, RNase H activity, or DNA/RNAhybrid-binding activity) or enzyme properties (e.g., thermostability,processivity, or fidelity). Such variants may be available in the art inthe public domain, available commercially, or may be made using knownmethods of mutagenesis, including directed evolutionary processes (e.g.,PACE or PANCE).

In various embodiments, the reverse transcriptase may be a variantreverse transcriptase. As used herein, a “variant reverse transcriptase”includes any naturally occurring or genetically engineered variantcomprising one or more mutations (including singular mutations,inversions, deletions, insertions, and rearrangements) relative to areference sequences (e.g., a reference wild type sequence). RT naturallyhave several activities, including an RNA-dependent DNA polymeraseactivity, ribonuclease H activity, and DNA-dependent DNA polymeraseactivity. Collectively, these activities enable the enzyme to convertsingle-stranded RNA into double-stranded cDNA. In retroviruses andretrotransposons, this cDNA can then integrate into the host genome,from which new RNA copies can be made via host-cell transcription.Variant RT's may comprise a mutation which impacts one or more of theseactivities (either which reduces or increases these activities, or whicheliminates these activities all together). In addition, variant RTs maycomprise one or more mutations which render the RT more or less stable,less prone to aggregation, and facilitates purification and/ordetection, and/or other the modification of properties orcharacteristics.

A person of ordinary skill in the art will recognize that variantreverse transcriptases derived from other reverse transcriptases,including but not limited to Moloney Murine Leukemia Virus (M-MLV);Human Immunodeficiency Virus (HIV) reverse transcriptase and avianSarcoma-Leukosis Virus (ASLV) reverse transcriptase, which includes butis not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, AvianMyeloblastosis Virus (AMV) reverse transcriptase, Avian ErythroblastosisVirus (AEV) Helper Virus MCAV reverse transcriptase, AvianMyelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase,Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reversetranscriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reversetranscriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reversetranscriptase, Rous Associated Virus (RAV) reverse transcriptase, andMyeloblastosis Associated Virus (MAV) reverse transcriptase may besuitably used in the subject methods and composition described herein.

One method of preparing variant RTs is by genetic modification (e.g., bymodifying the DNA sequence of a wild-type reverse transcriptase). Anumber of methods are known in the art that permit the random as well astargeted mutation of DNA sequences (see for example, Ausubel et. al.Short Protocols in Molecular Biology (1995) 3.sup.rd Ed. John Wiley &Sons, Inc.). In addition, there are a number of commercially availablekits for site-directed mutagenesis, including both conventional andPCR-based methods. Examples include the QuikChange Site-DirectedMutagenesis Kits (AGILENT®), the Q5® Site-Directed Mutagenesis Kit (NEWENGLAND BIOLABS®), and GeneArt™ Site-Directed Mutagenesis System(THERMOFISHER SCIENTIFIC®).

In addition, mutant reverse transcriptases may be generated byinsertional mutation or truncation (N-terminal, internal, or C-terminalinsertions or truncations) according to methodologies known to oneskilled in the art. The term “mutation,” as used herein, refers to asubstitution of a residue within a sequence, e.g., a nucleic acid oramino acid sequence, with another residue, or a deletion or insertion ofone or more residues within a sequence. Mutations are typicallydescribed herein by identifying the original residue followed by theposition of the residue within the sequence and by the identity of thenewly substituted residue. Various methods for making the amino acidsubstitutions (mutations) provided herein are well known in the art, andare provided by, for example, Green and Sambrook, Molecular Cloning: ALaboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2012)). Mutations can include a variety ofcategories, such as single base polymorphisms, microduplication regions,indel, and inversions, and is not meant to be limiting in any way.Mutations can include “loss-of-function” mutations which is the normalresult of a mutation that reduces or abolishes a protein activity. Mostloss-of-function mutations are recessive, because in a heterozygote thesecond chromosome copy carries an unmutated version of the gene codingfor a fully functional protein whose presence compensates for the effectof the mutation. Mutations also embrace “gain-of-function” mutations,which is one which confers an abnormal activity on a protein or cellthat is otherwise not present in a normal condition. Manygain-of-function mutations are in regulatory sequences rather than incoding regions, and can therefore have a number of consequences. Forexample, a mutation might lead to one or more genes being expressed inthe wrong tissues, these tissues gaining functions that they normallylack. Because of their nature, gain-of-function mutations are usuallydominant.

Older methods of site-directed mutagenesis known in the art rely onsub-cloning of the sequence to be mutated into a vector, such as an M13bacteriophage vector, that allows the isolation of single-stranded DNAtemplate. In these methods, one anneals a mutagenic primer (i.e., aprimer capable of annealing to the site to be mutated but bearing one ormore mismatched nucleotides at the site to be mutated) to thesingle-stranded template and then polymerizes the complement of thetemplate starting from the 3′ end of the mutagenic primer. The resultingduplexes are then transformed into host bacteria and plaques arescreened for the desired mutation.

More recently, site-directed mutagenesis has employed PCR methodologies,which have the advantage of not requiring a single-stranded template. Inaddition, methods have been developed that do not require sub-cloning.Several issues must be considered when PCR-based site-directedmutagenesis is performed. First, in these methods it is desirable toreduce the number of PCR cycles to prevent expansion of undesiredmutations introduced by the polymerase. Second, a selection must beemployed in order to reduce the number of non-mutated parental moleculespersisting in the reaction. Third, an extended-length PCR method ispreferred in order to allow the use of a single PCR primer set. Andfourth, because of the non-template-dependent terminal extensionactivity of some thermostable polymerases it is often necessary toincorporate an end-polishing step into the procedure prior to blunt-endligation of the PCR-generated mutant product.

Methods of random mutagenesis, which will result in a panel of mutantsbearing one or more randomly situated mutations, exist in the art. Sucha panel of mutants may then be screened for those exhibiting the desiredproperties, for example, increased stability, relative to a wild-typereverse transcriptase.

An example of a method for random mutagenesis is the so-called“error-prone PCR method.” As the name implies, the method amplifies agiven sequence under conditions in which the DNA polymerase does notsupport high fidelity incorporation. Although the conditions encouragingerror-prone incorporation for different DNA polymerases vary, oneskilled in the art may determine such conditions for a given enzyme. Akey variable for many DNA polymerases in the fidelity of amplificationis, for example, the type and concentration of divalent metal ion in thebuffer. The use of manganese ion and/or variation of the magnesium ormanganese ion concentration may therefore be applied to influence theerror rate of the polymerase.

In various aspects, the RT of the prime editors may be an “error-prone”reverse transcriptase variant. Error-prone reverse transcriptases thatare known and/or available in the art may be used. It will beappreciated that reverse transcriptases naturally do not have anyproofreading function; thus the error rate of reverse transcriptase isgenerally higher than DNA polymerases comprising a proofreadingactivity. The error-rate of any particular reverse transcriptase is aproperty of the enzyme's “fidelity,” which represents the accuracy oftemplate-directed polymerization of DNA against its RNA template. An RTwith high fidelity has a low-error rate. Conversely, an RT with lowfidelity has a high-error rate. The fidelity of M-MLV-based reversetranscriptases are reported to have an error rate in the range of oneerror in 15,000 to 27,000 nucleotides synthesized. See Boutabout et al.,“DNA synthesis fidelity by the reverse transcriptase of the yeastretrotransposon Ty1,” Nucleic Acids Res, 2001, 29: 2217-2222, which isincorporated by reference. Thus, for purposes of this application, thosereverse transcriptases considered to be “error-prone” or which areconsidered to have an “error-prone fidelity” are those having an errorrate that is less than one error in 15,000 nucleotides synthesized.

Error-prone reverse transcriptase also may be created throughmutagenesis of a starting RT enzyme (e.g., a wild type M-MLV RT). Themethod of mutagenesis is not limited and may include directed evolutionprocesses, such as phage-assisted continuous evolution (PACE) orphage-assisted noncontinuous evolution (PANCE). The term “phage-assistedcontinuous evolution (PACE),” as used herein, refers to continuousevolution that employs phage as viral vectors. The general concept ofPACE technology has been described, for example, in International PCTapplication, PCT/US2009/056194, filed Sep. 8, 2009, published as WO2010/028347 on Mar. 11, 2010; International PCT application,PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 onJun. 28, 2012; U.S. application, U.S. Pat. No. 9,023,594, issued May 5,2015, International PCT application, PCT/US2015/012022, filed Jan. 20,2015, published as WO 2015/134121 on Sep. 11, 2015, and InternationalPCT application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO2016/168631 on Oct. 20, 2016, the entire contents of each of which areincorporated herein by reference.

Error-prone reverse transcriptases may also be obtain by phage-assistednon-continuous evolution (PANCE),” which as used herein, refers tonon-continuous evolution that employs phage as viral vectors. PANCE is asimplified technique for rapid in vivo directed evolution using serialflask transfers of evolving ‘selection phage’ (SP), which contain a geneof interest to be evolved, across fresh E. coli host cells, therebyallowing genes inside the host E. coli to be held constant while genescontained in the SP continuously evolve. Serial flask transfers havelong served as a widely-accessible approach for laboratory evolution ofmicrobes, and, more recently, analogous approaches have been developedfor bacteriophage evolution. The PANCE system features lower stringencythan the PACE system.

Other error-prone reverse transcriptases have been described in theliterature, each of which are contemplated for use in the herein methodsand compositions. For example, error-prone reverse transcriptases havebeen described in Bebenek et al., “Error-prone Polymerization by HIV-1Reverse Transcriptase,” J Biol Chem, 1993, Vol. 268: 10324-10334 andSebastian-Martin et al., “Transcriptional inaccuracy thresholdattenuates differences in RNA-dependent DNA synthesis fidelity betweenretroviral reverse transcriptases,” Scientific Reports, 2018, Vol. 8:627, each of which are incorporated by reference. Still further, reversetranscriptases, including error-prone reverse transcriptases can beobtained from a commercial supplier, including ProtoScript® (II) ReverseTranscriptase, AMV Reverse Transcriptase, WarmStart® ReverseTranscriptase, and M-MuLV Reverse Transcriptase, all from NEW ENGLANDBIOLABS®, or AMV Reverse Transcriptase XL, SMARTScribe ReverseTranscriptase, GPR ultra-pure MMLV Reverse Transcriptase, all fromTAKARA BIO USA, INC. (formerly CLONTECH).

The herein disclosure also contemplates reverse transcriptases havingmutations in RNaseH domain. As mentioned above, one of the intrinsicproperties of reverse transcriptases is the RNase H activity, whichcleaves the RNA template of the RNA:cDNA hybrid concurrently withpolymerization. The RNase H activity can be undesirable for synthesis oflong cDNAs because the RNA template may be degraded before completion offull-length reverse transcription. The RNase H activity may also lowerreverse transcription efficiency, presumably due to its competition withthe polymerase activity of the enzyme. Thus, the present disclosurecontemplates any reverse transcriptase variants that comprise a modifiedRNaseH activity.

The herein disclosure also contemplates reverse transcriptases havingmutations in the RNA-dependent DNA polymerase domain. As mentionedabove, one of the intrinsic properties of reverse transcriptases is theRNA-dependent DNA polymerase activity, which incorporates thenucleobases into the nascent cDNA strand as coded by the template RNAstrand of the RNA:cDNA hybrid. The RNA-dependent DNA polymerase activitycan be increased or decreased (i.e., in terms of its rate ofincorporation) to either increase or decrease the processivity of theenzyme. Thus, the present disclosure contemplates any reversetranscriptase variants that comprise a modified RNA-dependent DNApolymerase activity such that the processivity of the enzyme of eitherincreased or decreased relative to an unmodified version.

Also contemplated herein are reverse transcriptase variants that havealtered thermostability characteristics. The ability of a reversetranscriptase to withstand high temperatures is an important aspect ofcDNA synthesis. Elevated reaction temperatures help denature RNA withstrong secondary structures and/or high GC content, allowing reversetranscriptases to read through the sequence. As a result, reversetranscription at higher temperatures enables full-length cDNA synthesisand higher yields, which can lead to an improved generation of the 3′flap ssDNA as a result of the prime editing process. Wild type M-MLVreverse transcriptase typically has an optimal temperature in the rangeof 37-48° C.; however, mutations may be introduced that allow for thereverse transcription activity at higher temperatures of over 48° C.,including 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56°C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65°C., 66° C., and higher.

The variant reverse transcriptases contemplated herein, includingerror-prone RTs, thermostable RTs, increase-processivity RTs, can beengineered by various routine strategies, including mutagenesis orevolutionary processes. In some cases, the variants can be produced byintroducing a single mutation. In other cases, the variants may requiremore than one mutation. For those mutants comprising more than onemutation, the effect of a given mutation may be evaluated byintroduction of the identified mutation to the wild-type gene bysite-directed mutagenesis in isolation from the other mutations borne bythe particular mutant. Screening assays of the single mutant thusproduced will then allow the determination of the effect of thatmutation alone.

Variant RT enzymes used herein may also include other “RT variants”having at least about 70% identical, at least about 80% identical, atleast about 90% identical, at least about 95% identical, at least about96% identical, at least about 97% identical, at least about 98%identical, at least about 99% identical, at least about 99.5% identical,or at least about 99.9% identical to any reference RT protein, includingany wild type RT, or mutant RT, or fragment RT, or other variant of RTdisclosed or contemplated herein or known in the art.

In some embodiments, an RT variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, or up to 100, or up to 200, or up to 300, or up to400, or up to 500 or more amino acid changes compared to a reference RT.In some embodiments, the RT variant comprises a fragment of a referenceRT, such that the fragment is at least about 70% identical, at leastabout 80% identical, at least about 90% identical, at least about 95%identical, at least about 96% identical, at least about 97% identical,at least about 98% identical, at least about 99% identical, at leastabout 99.5% identical, or at least about 99.9% identical to thecorresponding fragment of the reference RT. In some embodiments, thefragment is at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%identical, at least 96%, at least 97%, at least 98%, at least 99%, or atleast 99.5% of the amino acid length of a corresponding wild type RT(M-MLV reverse transcriptase) (e.g., SEQ ID NO: 89) or to any of thereverse transcriptases of SEQ ID NOs: 90-100.

In some embodiments, the disclosure also may utilize RT fragments whichretain their functionality and which are fragments of any hereindisclosed RT proteins. In some embodiments, the RT fragment is at least100 amino acids in length. In some embodiments, the fragment is at least100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or up to 600 or moreamino acids in length.

In still other embodiments, the disclosure also may utilize RT variantswhich are truncated at the N-terminus or the C-terminus, or both, by acertain number of amino acids which results in a truncated variant whichstill retains sufficient polymerase function. In some embodiments, theRT truncated variant has a truncation of at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids at theN-terminal end of the protein. In other embodiments, the RT truncatedvariant has a truncation of at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, or 250 amino acids at the C-terminal end of theprotein. In still other embodiments, the RT truncated variant has atruncation at the N-terminal and the C-terminal end which are the sameor different lengths.

For example, the prime editors disclosed herein may include a truncatedversion of M-MLV reverse transcriptase. In this embodiment, the reversetranscriptase contains 4 mutations (D200N, T306K, W313F, T330P; notingthat the L603W mutation present in PE2 is no longer present due to thetruncation). The DNA sequence encoding this truncated editor is 522 bpsmaller than PE2, and therefore makes its potentially useful forapplications where delivery of the DNA sequence is challenging due toits size (i.e., adeno-associated virus and lentivirus delivery). Thisembodiment is referred to as MMLV-RT(trunc) and has the following aminoacid sequence:

MMLV- TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQ RT(TRUNC)APLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDNSRLI N (SEQ ID NO: 766)

In various embodiments, the prime editors disclosed herein may compriseone of the RT variants described herein, or a RT variant thereof havingat least about 70% identical, at least about 80% identical, at leastabout 90% identical, at least about 95% identical, at least about 96%identical, at least about 97% identical, at least about 98% identical,at least about 99% identical, at least about 99.5% identical, or atleast about 99.9% identical to any reference Cas9 variants.

In still other embodiments, the present methods and compositions mayutilize a DNA polymerase that has been evolved into a reversetranscriptase, as described in Effefson et al., “Synthetic evolutionaryorigin of a proofreading reverse transcriptase,” Science, Jun. 24, 2016,Vol. 352: 1590-1593, the contents of which are incorporated herein byreference.

In certain other embodiments, the reverse transcriptase is provided as acomponent of a fusion protein also comprising a napDNAbp. In otherwords, in some embodiments, the reverse transcriptase is fused to anapDNAbp as a fusion protein.

In various embodiments, variant reverse transcriptases can be engineeredfrom wild type M-MLV reverse transcriptase as represented by SEQ ID NO:89.

In various embodiments, the prime editors described herein (with RTprovided as either a fusion partner or in trans) can include a variantRT comprising one or more of the following mutations: P51L, S67K, E69K,L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F,T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, orD653N in the wild type M-MLV RT of SEQ ID NO: 89 or at a correspondingamino acid position in another wild type RT polypeptide sequence.

Some exemplary reverse transcriptases that can be fused to napDNAbpproteins or provided as individual proteins according to variousembodiments of this disclosure are provided below. Exemplary reversetranscriptases include variants with at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity to thefollowing wild-type enzymes or partial enzymes:

Description Sequence (variant substitutions relative to wild type)Reverse TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP transcriptaseLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL (M-MLV RT)LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY wild typeTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN moloneySPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL murineLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM leukemiaGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWG virusPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL Used in PE1GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP (prime editorLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL 1 fusionNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS proteinSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA disclosedEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLK herein)ALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 700) M-MLV RTTLNIEDEYRLHETS KEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 701) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 702) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 740) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQ K ARLGIKPHIQRLLDQGILVPCQSPWNTP T330PLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQW L603WYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK E69K NSPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 703) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN E302R SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM GQPTPKTPRQLR RFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENS SP(SEQ ID NO: 704) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN E607K SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTS K GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 705) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN L139P SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 706) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN L435G SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LVI GAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 707) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN N454K SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LVILAPHAVEALVKQPPDRWLS KARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 708) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN T306K SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM GQPTPKTPRQLREFLG KAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 709) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN W313F SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM GQPTPKTPRQLREFLGTAGFCRL FIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 710) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN D524G SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL E562QLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM D583NGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT G GSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRA Q LIALTQALKMA EGKKLNVYT NSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 711) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN E302R SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL W313FLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM GQPTPKTPRQLR R FLGTAGFCRLF IPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 712) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN E607K SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL L139PLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTS K GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 713) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP P51L S67K LII LLKATSTPVSIKQYPM K QEARLGIKPHIQRLLDQGILVPCQSPWNTP T197ALLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQW H204RYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK E302K NSP A LFDEAL RRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTR F309NALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKET W313F VMGQPTPKTPRQLR KFLGTAG N CRL F IPGFAEMAAPLYPLTK P GTLFN T330PWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLT L435GQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM N454KGQPLVIGAPHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPV D524GVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT D583N GGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALK H594Q MAEGKKLNVYT NSRYAFATAHI Q GEIYRRRGLLTSEGKEIKNKDEILA D653NLLKALFLPKRLSIIHCPGHQKGHSAEARGNRMA N QAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 714) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200N P51L LII LLKATSTPVSIKQYPM K QEARLGIKPHIQRLLDQGILVPCQSWNTP S67K T197ALLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQW H204RYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK E302K NSP A LF N EAL RRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTR F309NALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKET W313F VMGQPTPKTPRQLR KFLGTAG N CRL F IPGFAEMAAPLYPLTK P GTLFN T330P L345GWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLT N454KQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM D524G GQPLVI GAPHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPV D583NVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT H594Q GGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALK D653N MAEGKKLNVYT NSRYAFATAHI Q GEIYRRRGLLTSEGKEIKNKDEILA LLKALFLPKRLSIIHCPGHQKGHSAEARGNRMAN QAARKAAITETPDT STLLIENSSP (SEQ ID NO: 715) M-MLV RTTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP D200NLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL T330PLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY L603WTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN T306K SPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL W313FLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM in PE2 GQPTPKTPRQLREFLG KAGFCRL F IPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 716)

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising one or more of the following mutations: P51X,S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X,W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X,E607X, or D653X in the wild type M-MLV RT of SEQ ID NO: 89 or at acorresponding amino acid position in another wild type RT polypeptidesequence, wherein “X” can be any amino acid.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a P51X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is L.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a S67X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is K.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a E69X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is K.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a L139X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is P.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a T197X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is A.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a D200X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is N.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a H204X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is R.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a F209X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is N.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a E302X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is K.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a E302X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is R.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a T306X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is K.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a F309X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is N.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a W313X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is F.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a T330X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is P.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a L345X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is G.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a L435X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is G.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a N454X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is K.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a D524X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is G.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a E562X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is Q.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a D583X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is N.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a H594X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is Q.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a L603X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is W.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a E607X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is K.

In various other embodiments, the prime editors described herein (withRT provided as either a fusion partner or in trans) can include avariant RT comprising a D653X mutation in the wild type M-MLV RT of SEQID NO: 89 or at a corresponding amino acid position in another wild typeRT polypeptide sequence, wherein “X” can be any amino acid. In certainembodiments, X is N.

Some exemplary reverse transcriptases that can be fused to napDNAbpproteins or provided as individual proteins according to variousembodiments of this disclosure are provided below. Exemplary reversetranscriptases include variants with at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% sequence identity to thefollowing wild-type enzymes or partial enzymes:

DESCRIPTION SEQUENCE (VARIANT SUBSTITUTIONS RELATIVE TO WILD TYPE)REVERSE TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL TRANSCRIPTASEIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL (M-MLV RT)PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT WILD TYPEVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP MOLONEYTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ MURINETLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ LEUKEMIAPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ VIRUSQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW USED INRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA PE1 (PRIMEPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL EDITOR 1PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG FUSIONQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN PROTEINVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKR DISCLOSEDLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP HEREIN) (SEQ ID NO: 89)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 106)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 107)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 108)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL E69KIIPLKATSTPVSIKQYPMSQ K ARLGIKPHIQRLLDQGILVPCQSPWNTPLL D200NPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT T330PVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP L603W TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 109)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP E302R TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ PTPKTPRQLR RFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 110)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP E607K TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTS K GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 111)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP L139P TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 112)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP L435G TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI G APHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 113)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP N454K TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA PHAVEALVKQPPDRWLS KARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 114)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP T306K TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ PTPKTPRQLREFLG KAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSS P(SEQ ID NO: 115)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP W313F TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ PTPKTPRQLREFLGTAGFCRL FIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 116)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP D524G TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ E562QTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ D583NPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT G GSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRA Q LIALTQALKMAEGKKLN VYT NSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 117)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP E302R TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ W313FTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ PTPKTPRQLR R FLGTAGFCRL FIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 118)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200NIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL T330PPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWYT L603WVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP E607K TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ L139PTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTS K GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 119)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL P51L S67K II LLKATSTPVSIKQYPM K QEARLGIKPHIQRLLDQGILVPCQSPWNTPL T197ALPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY H204RTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN E302K SP A LFDEAL RRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL F309NLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM W313F GQPTPKTPRQLR KFLGTAG N CRL F IPGFAEMAAPLYPLTK P GTLFNWGP T330PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG L435GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI N454KGAPHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPVVALNPA D524GTLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT G GSSLL D583NQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGK H594Q KLNVYT NSRYAFATAHI Q GEIYRRRGLLTSEGKEIKNKDEILALLKALFL D653NPKRLSIIHCPGHQKGHSAEARGNRMA N QAARKAAITETPDTSTLLIENS SP (SEQ ID NO: 120)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL P51L S67K II LLKATSTPVSIKQYPM K QEARLGIKPHIQRLLDQGILVPCQSPWNTPL T197ALPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY D200NTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN H204R SP A LF N EAL RRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL E302KLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM F309N GQPTPKTPRQLR KFLGTAG N CRL F IPGFAEMAAPLYPLTK P GTLFNWGP W313FDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG T330P L345GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI N454K GAPHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPVVALNPA D524GTLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT G GSSLL D583NQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGK H594Q KLNVYT NSRYAFATAHI Q GEIYRRRGLLTSEGKEIKNKDEILALLKALFL D653NPKRLSIIHCPGHQKGHSAEARGNRMA N QAARKAAITETPDTSTLLIENS SP (SEQ ID NO: 121)M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL D200N T330PIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL L603WPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT T306KVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP W313F TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ IN PE2TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ PTPKTPRQLREFLG K AGFCRL FIPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 122)

The prime editor (PE) system described here contemplates anypublicly-available reverse transcriptase described or disclosed in anyof the following U.S. patents (each of which are incorporated byreference in their entireties): U.S. Pat. Nos. 10,202,658; 10,189,831;10,150,955; 9,932,567; 9,783,791; 9,580,698; 9,534,201; and 9,458,484,and any variant thereof that can be made using known methods forinstalling mutations, or known methods for evolving proteins. Thefollowing references describe reverse transcriptases in art. Each oftheir disclosures are incorporated herein by reference in theirentireties.

-   Herzig, E., Voronin, N., Kucherenko, N. & Hizi, A. A Novel Leu92    Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in    Strand Transfer Causes a Loss of Viral Replication. J. Virol. 89,    8119-8129 (2015).-   Mohr, G. et al. A Reverse Transcriptase-Cas1 Fusion Protein Contains    a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer    Acquisition. Mol. Cell 72, 700-714.e8 (2018).-   Zhao, C., Liu, F. & Pyle, A. M. An ultraprocessive, accurate reverse    transcriptase encoded by a metazoan group II intron. RNA 24, 183-195    (2018).-   Zimmerly, S. & Wu, L. An Unexplored Diversity of Reverse    Transcriptases in Bacteria. Microbiol Spectr 3, MDNA3-0058-2014    (2015).-   Ostertag, E. M. & Kazazian Jr, H. H. Biology of Mammalian L1    Retrotransposons. Annual Review of Genetics 35, 501-538 (2001).-   Perach, M. & Hizi, A. Catalytic Features of the Recombinant Reverse    Transcriptase of Bovine Leukemia Virus Expressed in Bacteria.    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Cell 87, 905-916 (1996).-   Berkhout, B., Jebbink, M. & Zsíros, J. Identification of an Active    Reverse Transcriptase Enzyme Encoded by a Human Endogenous HERV-K    Retrovirus. Journal of Virology 73, 2365-2375 (1999).-   Kotewicz, M. L., Sampson, C. M., D'Alessio, J. M. & Gerard, G. F.    Isolation of cloned Moloney murine leukemia virus reverse    transcriptase lacking ribonuclease H activity. Nucleic Acids Res 16,    265-277 (1988).-   Arezi, B. & Hogrefe, H. Novel mutations in Moloney Murine Leukemia    Virus reverse transcriptase increase thermostability through tighter    binding to template-primer. Nucleic Acids Res 37, 473-481 (2009).-   Blain, S. W. & Goff, S. P. Nuclease activities of Moloney murine    leukemia virus reverse transcriptase. Mutants with altered substrate    specificities. J. Biol. Chem. 268, 23585-23592 (1993).-   Xiong, Y. & Eickbush, T. H. Origin and evolution of retroelements    based upon their reverse transcriptase sequences. EMBO J 9,    3353-3362 (1990).-   Herschhorn, A. & Hizi, A. Retroviral reverse transcriptases. Cell.    Mol. Life Sci. 67, 2717-2747 (2010).-   Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. Reverse    transcriptase of mouse mammary tumour virus: expression in bacteria,    purification and biochemical characterization. Biochem. J. 329 (Pt    3), 579-587 (1998).-   Liu, M. et al. Reverse Transcriptase-Mediated Tropism Switching in    Bordetella Bacteriophage. Science 295, 2091-2094 (2002).-   Luan, D. D., Korman, M. H., Jakubczak, J. L. & Eickbush, T. H.    Reverse transcription of R2Bm RNA is primed by a nick at the    chromosomal target site: a mechanism for non-LTR retrotransposition.    Cell 72, 595-605 (1993).-   Nottingham, R. M. et al. RNA-seq of human reference RNA samples    using a thermostable group II intron reverse transcriptase. RNA 22,    597-613 (2016).-   Telesnitsky, A. & Goff, S. P. RNase H domain mutations affect the    interaction between Moloney murine leukemia virus reverse    transcriptase and its primer-template. Proc. Natl. Acad. Sci. U.S.A.    90, 1276-1280 (1993).-   Halvas, E. K., Svarovskaia, E. S. & Pathak, V. K. Role of Murine    Leukemia Virus Reverse Transcriptase Deoxyribonucleoside    Triphosphate-Binding Site in Retroviral Replication and In Vivo    Fidelity. Journal of Virology 74, 10349-10358 (2000).-   Nowak, E. et al. Structural analysis of monomeric retroviral reverse    transcriptase in complex with an RNA/DNA hybrid. Nucleic Acids Res    41, 3874-3887 (2013).-   Stamos, J. L., Lentzsch, A. M. & Lambowitz, A. M. Structure of a    Thermostable Group II Intron Reverse Transcriptase with    Template-Primer and Its Functional and Evolutionary Implications.    Molecular Cell 68, 926-939.e4 (2017).-   Das, D. & Georgiadis, M. M. The Crystal Structure of the Monomeric    Reverse Transcriptase from Moloney Murine Leukemia Virus. Structure    12, 819-829 (2004).-   Avidan, O., Meer, M. E., Oz, I. & Hizi, A. The processivity and    fidelity of DNA synthesis exhibited by the reverse transcriptase of    bovine leukemia virus. European Journal of Biochemistry 269, 859-867    (2002).-   Gerard, G. F. et al. The role of template-primer in protection of    reverse transcriptase from thermal inactivation. Nucleic Acids Res    30, 3118-3129 (2002).-   Monot, C. et al. The Specificity and Flexibility of L1 Reverse    Transcription Priming at Imperfect T-Tracts. PLOS Genetics 9,    e1003499 (2013).-   Mohr, S. et al. Thermostable group II intron reverse transcriptase    fusion proteins and their use in cDNA synthesis and next-generation    RNA sequencing. RNA 19, 958-970 (2013).

Any of the references noted above which relate to reverse transcriptasesare hereby incorporated by reference in their entireties, if not alreadystated so.

[4] PE Fusion Proteins

The prime editor (PE) system described herein contemplate fusionproteins comprising a napDNAbp and a polymerase (e.g., DNA-dependent DNApolymerase or RNA-dependent DNA polymerase, such as, reversetranscriptase), and optionally joined by a linker. The applicationcontemplates any suitable napDNAbp and polymerase (e.g., DNA-dependentDNA polymerase or RNA-dependent DNA polymerase, such as, reversetranscriptase) to be combined in a single fusion protein. Examples ofnapDNAbps and polymerases (e.g., DNA-dependent DNA polymerase orRNA-dependent DNA polymerase, such as, reverse transcriptase) are eachdefined herein. Since polymerases are well-known in the art, and theamino acid sequences are readily available, this disclosure is not meantin any way to be limited to those specific polymerases identifiedherein.

In various embodiments, the fusion proteins may comprise any suitablestructural configuration. For example, the fusion protein may comprisefrom the N-terminus to the C-terminus direction, a napDNAbp fused to apolymerase (e.g., DNA-dependent DNA polymerase or RNA-dependent DNApolymerase, such as, reverse transcriptase). In other embodiments, thefusion protein may comprise from the N-terminus to the C-terminusdirection, a polymerase (e.g., a reverse transcriptase) fused to anapDNAbp. The fused domain may optionally be joined by a linker, e.g.,an amino acid sequence. In other embodiments, the fusion proteins maycomprise the structure NH₂-[napDNAbp]-[polymerase]-COOH; orNH₂-[polymerase]-[napDNAbp]-COOH, wherein each instance of “]-[”indicates the presence of an optional linker sequence. In embodimentswherein the polymerase is a reverse transcriptase, the fusion proteinsmay comprise the structure NH₂-[napDNAbp]-[RT]-COOH; orNH₂-[RT]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates thepresence of an optional linker sequence.

An exemplary fusion protein is depicted in FIG. 14 , which shows afusion protein comprising an MLV reverse transcriptase (“MLV-RT”) fusedto a nickase Cas9 (“Cas9(H840A)”) via a linker sequence. This example isnot intended to limit scope of fusion proteins that may be utilized forthe prime editor (PE) system described herein.

In various embodiments, the prime editor fusion protein may have thefollowing amino acid sequence (referred to herein as “PE1”), whichincludes a Cas9 variant comprising an H840A mutation (i.e., a Cas9nickase) and an M-MLV RT wild type, as well as an N-terminal NLSsequence (19 amino acids) and an amino acid linker (32 amino acids) thatjoins the C-terminus of the Cas9 nickase domain to the N-terminus of theRT domain. The PE1 fusion protein has the following structure:[NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]. The amino acid sequence ofPE1 and its individual components are as follows:

DESCRIPTION SEQUENCE PE1 FUSION MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPS PROTEINKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR CAS9(H840A)-KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI MMLV_RT(WT)VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS QLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSS PSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 123) KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ ID NO: 124),BOTTOM:(SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 126)33-AMINO ACID LINKER  (SEQ ID NO: 127)M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 128) PE1-N-MKRTADGSEFESPKKKRKV (SEQ ID NO: 124) TERMINAL NLS PE1-CAS9DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA (H840A)LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH (METRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK MINUS)ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVD AIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD (SEQ ID NO: 130)PE1- SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127) LINKER BETWEENCAS9 DOMAIN AND RT DOMAIN (33 AMINO ACIDS) PE-M-TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL MLV RTIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 132) PE1-C-SGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 133) TERMINAL NLS

In another embodiment, the prime editor fusion protein may have thefollowing amino acid sequence (referred to herein as “PE2”), whichincludes a Cas9 variant comprising an H840A mutation (i.e., a Cas9nickase) and an M-MLV RT comprising mutations D200N, T330P, L603W,T306K, and W313F, as well as an N-terminal NLS sequence (19 amino acids)and an amino acid linker (33 amino acids) that joins the C-terminus ofthe Cas9 nickase domain to the N-terminus of the RT domain. The PE2fusion protein has the following structure:[NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)].The amino acid sequence of PE2 is as follows:

PE2 FUSION MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPS PROTEINKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR CAS9(H840A)-KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI MMLV_RTVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF D200NLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR T330PLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA L603WKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV T306KNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ W313FSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS QLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENS SPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 134) KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ ID NO: 124),BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 137)33-AMINO ACID LINKER  (SEQ ID NO: 127)M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 139) PE2-N-MKRTADGSEFESPKKKRKV (SEQ ID NO: 124) TERMINAL NLS PE2-CAS9DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA (H840A)LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH (METRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK MINUS)ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVD AIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD (SEQ ID NO: 141)PE2- SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127) LINKER BETWEENCAS9 DOMAIN AND RT DOMAIN (33 AMINO ACIDS) PE2-TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL MMLV_RTIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL D200NPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT T330PVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP L603W TLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ T306KTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ W313F PTPKTPRQLREFLG KAGFCRL F IPGFAEMAAPLYPLTK P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 143) PE2-C-SGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 133) TERMINAL NLS

In still other embodiments, the prime editor fusion protein may have thefollowing amino acid sequences:

PE FUSION PROTEIN MMLV_RT(WT)-32AA-CAS9(H840A) (SEQ ID NO: 145)MKRTADGSEFESPKKKRKV TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSS DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD SGGSKRTADGSEFEPKKKRKVKEY: NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124), BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 147)33-AMINO ACID LINKER  (SEQ ID NO: 127)M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 149)PE FUSION PROTEIN MMLV_RT(WT)-60AA-CAS9(H840A) (SEQ ID NO: 150)MKRTADGSEFESPKKKRKV TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSG GSSGGSDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSKRTADGSEFEPKKKRKV KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124), BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 153)AMINO ACID LINKER  (SEQ ID NO: 175)M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 149)PE FUSION PROTEIN CAS9(H840A)-FEN1-MMLV_RT D200N T330P L603W T306K W313F(SEQ ID NO: 154) MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS QLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS GIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKRGK SGGSSGGSSGSETPGTSES ATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPRGHQKGHSAEARGNMADQAARKAAITETPDTSTLLIENSSP SGGSKRTAD GSEFEPKKKRKV KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124), BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 157)33-AMINO ACID LINKER 1  (SEQ ID NO: 127)M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 159) 33-AMINO ACID LINKER 2 (SEQ ID NO: 127) FEN1 (SEQ ID NO: 161)

In other embodiments, the prime editor fusion proteins can be based onSaCas9 or on SpCas9 nickases with altered PAM specificities, such as thefollowing exemplary sequences:

SACAS9-M-MLV MKRTADGSEFESPKKKRKVGKRNYILGLDIGITSVGYGIIDYETRRT PRIME EDITOR DVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKR KV (SEQ ID NO: 162)SPCAS9(H840A)- MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY VRQR-MALONEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR MURINERRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH LEUKEMIA VIRUSERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL REVERSEAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN TRANSCRIPTASEASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL PRIME EDITORGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 163) SPCAS9(H840A)-MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY VRER-MALONEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR MURINERRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH LEUKEMIA VIRUSERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL REVERSEAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN TRANSCRIPTASEASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL PRIME EDITORGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 164)

In yet other embodiments, the prime editor fusion proteins contemplatedherein may include a Cas9 nickase (e.g., Cas9 (H840A)) fused to atruncated version of M-MLV reverse transcriptase. In this embodiment,the reverse transcriptase also contains 4 mutations (D200N, T306K,W313F, T330P; noting that the L603W mutation present in PE2 is no longerpresent due to the truncation). The DNA sequence encoding this truncatededitor is 522 bp smaller than PE2, and therefore makes its potentiallyuseful for applications where delivery of the DNA sequence ischallenging due to its size (i.e. adeno-associated virus and lentivirusdelivery). This embodiment is referred to as Cas9(H840A)-MMLV-RT(trunc)or “PE2-short” or “PE2-trunc” and has the following amino acid sequence:

CAS9(H840A)- MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDE MMLV-YKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK RT(TRUNC) ORRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLV PE2-SHORTEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSE SATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLP EEGLQHNCLDNSRLINSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 765) KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ ID NO:124), BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 157)33-AMINO ACID LINKER 1  (SEQ ID NO: 127)M-MLV TRUNCATED REVERSE TRANSCRIPTASE (SEQ ID NO: 766)

See FIG. 75 , which provides a bar graph comparing the efficiency (i.e.,“% of total sequencing reads with the specified edit or indels”) of PE2,PE2-trunc, PE3, and PE3-trunc over different target sites in variouscell lines. The data shows that the prime editors comprising thetruncated RT variants were about as efficient as the prime editorscomprising the non-truncated RT proteins.

In various embodiments, the prime editor fusion proteins contemplatedherein may also include any variants of the above-disclosed sequenceshaving an amino acid sequence that is at least about 70% identical, atleast about 80% identical, at least about 90% identical, at least about95% identical, at least about 96% identical, at least about 97%identical, at least about 98% identical, at least about 99% identical,at least about 99.5% identical, or at least about 99.9% identical toPE1, PE2, or any of the above indicated prime editor fusion sequences.

In certain embodiments, linkers may be used to link any of the peptidesor peptide domains or moieties of the invention (e.g., a napDNAbp linkedor fused to a reverse transcriptase).

[5] Linkers and Other Domains

The PE fusion proteins may comprise various other domains besides thenapDNAbp (e.g., Cas9 domain) and the polymerase domain (e.g., RTdomain). For example, in the case where the napDNAbp is a Cas9 and thepolymerase is a RT, the PE fusion proteins may comprise one or morelinkers that join the Cas9 domain with the RT domain. The linkers mayalso join other functional domains, such as nuclear localizationsequences (NLS) or a FEN1 (or other flap endonuclease) to the PE fusionproteins or a domain thereof.

In addition, in embodiments involving trans prime editing, linkers maybe used to link tPERT recruitment protein to a prime editor, e.g.,between the tPERt recruitment protein and the napDNAbp. See e.g., FIG.3G for an exemplary schematic of a trans prime editor (tPE) thatincludes linkers to separately fuse a polymerase domain and a recruitingprotein domain to a napDNAbp.

A. Linkers

As defined above, the term “linker,” as used herein, refers to achemical group or a molecule linking two molecules or moieties, e.g., abinding domain and a cleavage domain of a nuclease. In some embodiments,a linker joins a gRNA binding domain of an RNA-programmable nuclease andthe catalytic domain of a polymerase (e.g., a reverse transcriptase). Insome embodiments, a linker joins a dCas9 and reverse transcriptase.Typically, the linker is positioned between, or flanked by, two groups,molecules, or other moieties and connected to each one via a covalentbond, thus connecting the two. In some embodiments, the linker is anamino acid or a plurality of amino acids (e.g., a peptide or protein).In some embodiments, the linker is an organic molecule, group, polymer,or chemical moiety. In some embodiments, the linker is 5-100 amino acidsin length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45,45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 aminoacids in length. Longer or shorter linkers are also contemplated.

The linker may be as simple as a covalent bond, or it may be a polymericlinker many atoms in length. In certain embodiments, the linker is apolypeptide or based on amino acids. In other embodiments, the linker isnot peptide-like. In certain embodiments, the linker is a covalent bond(e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond,etc.). In certain embodiments, the linker is a carbon-nitrogen bond ofan amide linkage. In certain embodiments, the linker is a cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphaticor heteroaliphatic linker. In certain embodiments, the linker ispolymeric (e.g., polyethylene, polyethylene glycol, polyamide,polyester, etc.). In certain embodiments, the linker comprises amonomer, dimer, or polymer of aminoalkanoic acid. In certainembodiments, the linker comprises an aminoalkanoic acid (e.g., glycine,ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid,4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments,the linker comprises a monomer, dimer, or polymer of aminohexanoic acid(Ahx). In certain embodiments, the linker is based on a carbocyclicmoiety (e.g., cyclopentane, cyclohexane). In other embodiments, thelinker comprises a polyethylene glycol moiety (PEG). In otherembodiments, the linker comprises amino acids. In certain embodiments,the linker comprises a peptide. In certain embodiments, the linkercomprises an aryl or heteroaryl moiety. In certain embodiments, thelinker is based on a phenyl ring. The linker may included functionalizedmoieties to facilitate attachment of a nucleophile (e.g., thiol, amino)from the peptide to the linker. Any electrophile may be used as part ofthe linker. Exemplary electrophiles include, but are not limited to,activated esters, activated amides, Michael acceptors, alkyl halides,aryl halides, acyl halides, and isothiocyanates.

In some other embodiments, the linker comprises the amino acid sequence(GGGGS)_(n) (SEQ ID NO: 165), (G)_(n) (SEQ ID NO: 166), (EAAAK)_(n) (SEQID NO: 167), (GGS)_(n) (SEQ ID NO: 168), (SGGS)_(n) (SEQ ID NO: 169),(XP). (SEQ ID NO: 170), or any combination thereof, wherein n isindependently an integer between 1 and 30, and wherein X is any aminoacid. In some embodiments, the linker comprises the amino acid sequence(GGS)N(SEQ ID NO: 176), wherein n is 1, 3, or 7. In some embodiments,the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ IDNO: 171). In some embodiments, the linker comprises the amino acidsequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 172). In someembodiments, the linker comprises the amino acid sequence SGGSGGSGGS(SEQ ID NO: 173). In some embodiments, the linker comprises the aminoacid sequence SGGS (SEQ ID NO: 174). In other embodiments, the linkercomprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSG GS (SEQ IDNO: 175, 60AA).

In certain embodiments, linkers may be used to link any of the peptidesor peptide domains or moieties of the invention (e.g., a napDNAbp linkedor fused to a reverse transcriptase).

As defined above, the term “linker,” as used herein, refers to achemical group or a molecule linking two molecules or moieties, e.g., abinding domain and a cleavage domain of a nuclease. In some embodiments,a linker joins a gRNA binding domain of an RNA-programmable nuclease andthe catalytic domain of a recombinase. In some embodiments, a linkerjoins a dCas9 and reverse transcriptase. Typically, the linker ispositioned between, or flanked by, two groups, molecules, or othermoieties and connected to each one via a covalent bond, thus connectingthe two. In some embodiments, the linker is an amino acid or a pluralityof amino acids (e.g., a peptide or protein). In some embodiments, thelinker is an organic molecule, group, polymer, or chemical moiety. Insome embodiments, the linker is 5-100 amino acids in length, forexample, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60,60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length.Longer or shorter linkers are also contemplated.

The linker may be as simple as a covalent bond, or it may be a polymericlinker many atoms in length. In certain embodiments, the linker is apolypeptide or based on amino acids. In other embodiments, the linker isnot peptide-like. In certain embodiments, the linker is a covalent bond(e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond,etc.). In certain embodiments, the linker is a carbon-nitrogen bond ofan amide linkage. In certain embodiments, the linker is a cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphaticor heteroaliphatic linker. In certain embodiments, the linker ispolymeric (e.g., polyethylene, polyethylene glycol, polyamide,polyester, etc.). In certain embodiments, the linker comprises amonomer, dimer, or polymer of aminoalkanoic acid. In certainembodiments, the linker comprises an aminoalkanoic acid (e.g., glycine,ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid,4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments,the linker comprises a monomer, dimer, or polymer of aminoHEXAnoic acid(Ahx). In certain embodiments, the linker is based on a carbocyclicmoiety (e.g., cyclopentane, cycloHEXAne). In other embodiments, thelinker comprises a polyethylene glycol moiety (PEG). In otherembodiments, the linker comprises amino acids. In certain embodiments,the linker comprises a peptide. In certain embodiments, the linkercomprises an aryl or heteroaryl moiety. In certain embodiments, thelinker is based on a phenyl ring. The linker may included functionalizedmoieties to facilitate attachment of a nucleophile (e.g., thiol, amino)from the peptide to the linker. Any electrophile may be used as part ofthe linker. Exemplary electrophiles include, but are not limited to,activated esters, activated amides, Michael acceptors, alkyl halides,aryl halides, acyl halides, and isothiocyanates.

In some other embodiments, the linker comprises the amino acid sequence(GGGGS)n (SEQ ID NO: 165), (G)n (SEQ ID NO: 166), (EAAAK)_(n) (SEQ IDNO: 167), (GGS)_(n) (SEQ ID NO: 168), (SGGS)n (SEQ ID NO: 169), (XP)n(SEQ ID NO: 170), or any combination thereof, wherein n is independentlyan integer between 1 and 30, and wherein X is any amino acid. In someembodiments, the linker comprises the amino acid sequence (GGS)N(SEQ IDNO: 176), wherein n is 1, 3, or 7. In some embodiments, the linkercomprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 171). Insome embodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 172). In some embodiments,the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO:173). In some embodiments, the linker comprises the amino acid sequenceSGGS (SEQ ID NO: 174).

In particular, the following linkers can be used in various embodimentsto join prime editor domains with one another:

(SEQ ID NO: 767) GGS; (SEQ ID NO: 768) GGSGGS; (SEQ ID NO: 769)GGSGGSGGS; (SEQ ID NO: 127) SGGSSGGSSGSETPGTSESATPESSGGSSGGSS;(SEQ ID NO: 171) SGSETPGTSESATPES; (SEQ ID NO: 175)SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDG SGSGGSSGGS.

B. Nuclear Localization Sequence (NLS)

In various embodiments, the PE fusion proteins may comprise one or morenuclear localization sequences (NLS), which help promote translocationof a protein into the cell nucleus. Such sequences are well-known in theart and can include the following examples:

DESCRIPTION SEQUENCE SEQ ID NO: NLS OF PKKKRKV SEQ ID NO: 16 SV40LARGE T- AG NLS MKRTADGSEFESPKKKRKV SEQ ID NO: 124 NLSMDSLLMNRRKFLYQFKNVRW SEQ ID NO: 17 AKGRRETYLC NLS OFAVKRPAATKKAGQAKKKKLD SEQ ID NO: 190 NUCLEOP LASMIN NLS OFMSRRRKANPTKLSENAKKLA SEQ ID NO: 191 EGL-13 KEVEN NLS OF C- PAAKRVKLDSEQ ID NO: 192 MYC NLS OF KLKIKRPVK SEQ ID NO: 193 TUS- PROTEIN NLS OFVSRKRPRP SEQ ID NO: 194 POLYOMA LARGE T- AG NLS OF EGAPPAKRARSEQ ID NO: 195 HEPATITIS D VIRUS ANTIGEN NLS OF PPQPKKKPLDGESEQ ID NO: 196 MURINE P53 NLS OF SGGSKRTADGSEFEPKKKRKV SEQ ID NO: 133PE1 AND PE2

The NLS examples above are non-limiting. The PE fusion proteins maycomprise any known NLS sequence, including any of those described inCokol et al., “Finding nuclear localization signals,” EMBO Rep., 2000,1(5): 411-415 and Freitas et al., “Mechanisms and Signals for theNuclear Import of Proteins,” Current Genomics, 2009, 10(8): 550-7, eachof which are incorporated herein by reference.

In various embodiments, the prime editors and constructs encoding theprime editors disclosed herein further comprise one or more, preferably,at least two nuclear localization signals. In certain embodiments, theprime editors comprise at least two NLSs. In embodiments with at leasttwo NLSs, the NLSs can be the same NLSs or they can be different NLSs.In addition, the NLSs may be expressed as part of a fusion protein withthe remaining portions of the prime editors. In some embodiments, one ormore of the NLSs are bipartite NLSs (“bpNLS”). In certain embodiments,the disclosed fusion proteins comprise two bipartite NLSs. In someembodiments, the disclosed fusion proteins comprise more than twobipartite NLSs.

The location of the NLS fusion can be at the N-terminus, the C-terminus,or within a sequence of a prime editor (e.g., inserted between theencoded napDNAbp component (e.g., Cas9) and a polymerase domain (e.g., areverse transcriptase domain).

The NLSs may be any known NLS sequence in the art. The NLSs may also beany future-discovered NLSs for nuclear localization. The NLSs also maybe any naturally-occurring NLS, or any non-naturally occurring NLS(e.g., an NLS with one or more desired mutations).

The term “nuclear localization sequence” or “NLS” refers to an aminoacid sequence that promotes import of a protein into the cell nucleus,for example, by nuclear transport. Nuclear localization sequences areknown in the art and would be apparent to the skilled artisan. Forexample, NLS sequences are described in Plank et al., International PCTapplication PCT/EP2000/01 1690, filed Nov. 23, 2000, published asWO/2001/038547 on May 31, 2001, the contents of which are incorporatedherein by reference. In some embodiments, an NLS comprises the aminoacid sequence PKKKRKV (SEQ ID NO: 16), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC(SEQ ID NO: 17), KRTADGSEFESPKKKRKV (SEQ ID NO: 3864), orKRTADGSEFEPKKKRKV (SEQ ID NO: 125). In other embodiments, NLS comprisesthe amino acid sequences NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 136), PAAKRVKLD(SEQ ID NO: 192), RQRRNELKRSF (SEQ ID NO: 3934),NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 3935).

In one aspect of the disclosure, a prime editor may be modified with oneor more nuclear localization signals (NLS), preferably at least twoNLSs. In certain embodiments, the prime editors are modified with two ormore NLSs. The disclosure contemplates the use of any nuclearlocalization signal known in the art at the time of the disclosure, orany nuclear localization signal that is identified or otherwise madeavailable in the state of the art after the time of the instant filing.A representative nuclear localization signal is a peptide sequence thatdirects the protein to the nucleus of the cell in which the sequence isexpressed. A nuclear localization signal is predominantly basic, can bepositioned almost anywhere in a protein's amino acid sequence, generallycomprises a short sequence of four amino acids (Autieri & Agrawal,(1998) J. Biol. Chem. 273: 14731-37, incorporated herein by reference)to eight amino acids, and is typically rich in lysine and arginineresidues (Magin et al., (2000) Virology 274: 11-16, incorporated hereinby reference). Nuclear localization signals often comprise prolineresidues. A variety of nuclear localization signals have been identifiedand have been used to effect transport of biological molecules from thecytoplasm to the nucleus of a cell. See, e.g., Tinland et al., (1992)Proc. Natl. Acad. Sci. U.S.A. 89:7442-46; Moede et al., (1999) FEBSLett. 461:229-34, which is incorporated by reference. Translocation iscurrently thought to involve nuclear pore proteins.

Most NLSs can be classified in three general groups: (i) a monopartiteNLS exemplified by the SV40 large T antigen NLS (PKKKRKV (SEQ ID NO:16)); (ii) a bipartite motif consisting of two basic domains separatedby a variable number of spacer amino acids and exemplified by theXenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL (SEQ ID NO: 3936)); and(iii) noncanonical sequences such as M9 of the hnRNP A1 protein, theinfluenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS(Dingwall and Laskey 1991).

Nuclear localization signals appear at various points in the amino acidsequences of proteins. NLS's have been identified at the N-terminus, theC-terminus and in the central region of proteins. Thus, the disclosureprovides prime editors that may be modified with one or more NLSs at theC-terminus, the N-terminus, as well as at in internal region of theprime editor. The residues of a longer sequence that do not function ascomponent NLS residues should be selected so as not to interfere, forexample tonically or sterically, with the nuclear localization signalitself. Therefore, although there are no strict limits on thecomposition of an NLS-comprising sequence, in practice, such a sequencecan be functionally limited in length and composition.

The present disclosure contemplates any suitable means by which tomodify a prime editor to include one or more NLSs. In one aspect, theprime editors may be engineered to express a prime editor protein thatis translationally fused at its N-terminus or its C-terminus (or both)to one or more NLSs, i.e., to form a prime editor-NLS fusion construct.In other embodiments, the prime editor-encoding nucleotide sequence maybe genetically modified to incorporate a reading frame that encodes oneor more NLSs in an internal region of the encoded prime editor. Inaddition, the NLSs may include various amino acid linkers or spacerregions encoded between the prime editor and the N-terminally,C-terminally, or internally-attached NLS amino acid sequence, e.g, andin the central region of proteins. Thus, the present disclosure alsoprovides for nucleotide constructs, vectors, and host cells forexpressing fusion proteins that comprise a prime editor and one or moreNLSs.

The prime editors described herein may also comprise nuclearlocalization signals which are linked to a prime editor through one ormore linkers, e.g., and polymeric, amino acid, nucleic acid,polysaccharide, chemical, or nucleic acid linker element. The linkerswithin the contemplated scope of the disclosure are not intended to haveany limitations and can be any suitable type of molecule (e.g., polymer,amino acid, polysaccharide, nucleic acid, lipid, or any syntheticchemical linker domain) and be joined to the prime editor by anysuitable strategy that effectuates forming a bond (e.g., covalentlinkage, hydrogen bonding) between the prime editor and the one or moreNLSs.

C. Flap Endonucleases (e.g., FEN1)

In various embodiments, the PE fusion proteins may comprise one or moreflap endonucleases (e.g., FEN1), which refers to an enzyme thatcatalyzes the removal of 5′ single strand DNA flaps. These are naturallyoccurring enzymes that process the removal of 5′ flaps formed duringcellular processes, including DNA replication. The prime editing methodsherein described may utilize endogenously supplied flap endonucleases orthose provided in trans to remove the 5′ flap of endogenous DNA formedat the target site during prime editing. Flap endonucleases are known inthe art and can be found described in Patel et al., “Flap endonucleasespass 5′-flaps through a flexible arch using a disorder-thread-ordermechanism to confer specificity for free 5′-ends,” Nucleic AcidsResearch, 2012, 40(10): 4507-4519 and Tsutakawa et al., “Human flapendonuclease structures, DNA double-base flipping, and a unifiedunderstanding of the FEN1 superfamily,” Cell, 2011, 145(2): 198-211(each of which are incorporated herein by reference). An exemplary flapendonuclease is FEN1, which can be represented by the following aminoacid sequence:

Description Sequence SEQ ID NO: FEN1MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI SEQ ID NO: Wild typeYQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME 198 (wt)NGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKR GK

The flap endonucleases may also include any FEN1 variant, mutant, orother flap endonuclease ortholog, homolog, or variant. Non-limiting FEN1variant examples are as follows:

Description Sequence SEQ ID NO: FEN1MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI SEQ ID NO: K168RYQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME 199 (relative toNGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQ FEN1 wt)AQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGI PYLDAPSEAEASCAALV RAGKVYAAATEDMDCLTFGS PVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKR GK FEN1MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI SEQ ID NO: S187AYQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME 200 (relative toNGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQ FEN1 wt)AQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFG APVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFK RGK FEN1MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI SEQ ID NO: K354RYQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME 201 (relative toNGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQ FEN1 wt)AQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLD DFFKVTGSLSSA RRKEPEPKGSTKKKAKTGAAGKFKR GK GEN1 MGVNDLWQILEPVKQHIPLRNLGGKTIAVDLSLWVCESEQ ID NO: AQTVKKMMGSVMKPHLRNLFFRISYLTQMDVKLVFV 202MEGEPPKLKADVISKRNQSRYGSSGKSWSQKTGRSHFKSVLRECLHMLECLGIPWVQAAGEAEAMCAYLNAGGHVDGCLTNDGDTFLYGAQTVYRNFTMNTKDPHVDCYTMSSIKSKLGLDRDALVGLAILLGCDYLPKGVPGVGKEQALKLIQILKGQSLLQRFNRWNETSCNSSPQLLVTKKLAHCSVCSHPGSPKDHERNGCRLCKSDKYCEPHDYEYCCPCEWHRTEHDRQLSEVENNIKKKACCCEGFPFHEVIQEFLLNKDKLVKVIRYQRPDLLLFQRFTLEKMEWPNHYACEKLLVLLTHYDMIERKLGSRNSNQLQPIRIVKTRIRNGVHCFEIEWEKPEHYAMEDKQHGEFALLTIEEESLFEAAYPEIVAVYQKQKLEIKGKKQKRIKPKENNLPEPDEVMSFQSHMTLKPTCEIFHKQNSKLNSGISPDPTLPQESISASLNSLLLPKNTPCLNAQEQFMSSLRPLAIQQIKAVSKSLISESSQPNTSSHNISVIADLHLSTIDWEGTSFSNSPAIQRNTFSHDLKSEVESELSAIPDGFENIPEQLSCESERYTANIKKVLDEDSDGISPEEHLLSGITDLCLQDLPLKERIFTKLSYPQDNLQPDVNLKTLSILSVKESCIANSGSDCTSHLSKDLPGIPLQNESRDSKILKGDQLLQEDYKVNTSVPYSVSNTVVKTCNVRPPNTALDHSRKVDMQTTRKILMKKSVCLDRHSSDEQSAPVFGKAKYTTQRMKHSSQKHNSSHFKESGHNKLSSPKIHIKETEQCVRSYETAENEESCFPDSTKSSLSSLQCHKKENNSGTCLDSPLPLRQRLKLRFQST ERCC5MGVQGLWKLLECSGRQVSPEALEGKILAVDISIWLNQ SEQ ID NO:ALKGVRDRHGNSIENPHLLTLFHRLCKLLFFRIRPIFVF 203DGDAPLLKKQTLVKRRQRKDLASSDSRKTTEKLLKTFLKRQAIKTAFRSKRDEALPSLTQVRRENDLYVLPPLQEEEKHSSEEEDEKEWQERMNQKQALQEEFFHNPQAIDIESEDFSSLPPEVKHEILTDMKEFTKRRRTLFEAMPEESDDFSQYQLKGLLKKNYLNQHIEHVQKEMNQQHSGHIRRQYEDEGGFLKEVESRRVVSEDTSHYILIKGIQAKTVAEVDSESLPSSSKMHGMSFDVKSSPCEKLKTEKEPDATPPSPRTLLAMQAALLGSSSEEELESENRRQARGRNAPAAVDEGSISPRTLSAIKRALDDDEDVKVCAGDDVQTGGPGAEEMRINSSTENSDEGLKVRDGKGIPFTATLASSSVNSAEEHVASTNEGREPTDSVPKEQMSLVHVGTEAFPISDESMIKDRKDRLPLESAVVRHSDAPGLPNGRELTPASPTCTNSVSKNETHAEVLEQQNELCPYESKFDSSLLSSDDETKCKPNSASEVIGPVSLQETSSIVSVPSEAVDNVENVVSFNAKEHENFLETIQEQQTTESAGQDLISIPKAVEPMEIDSEESESDGSFIEVQSVISDEELQAEFPETSKPPSEQGEEELVGTREGEAPAESESLLRDNSERDDVDGEPQEAEKDAEDSLHEWQDINLEELETLESNLLAQQNSLKAQKQQQERIAATVTGQMFLESQELLRLFGIPYIQAPMEAEAQCAILDLTDQTSGTITDDSDIWLFGARHVYRNFFNKNKFVEYYQYVDFHNQLGLDRNKLINLAYLLGSDYTEGIPTVGCVTAMEILNEFPGHGLEPLLKFSEWWHEAQKNPKIRPNPHDTKVKKKLRTLQLTPGFPNPAVAEAYLKPVVDDSKGSFLWGKPDLDKIREFCQRYFGWNRTKTDESLFPVLKQLDAQQTQLRIDSFFRLAQQEKEDAKRIKSQRLNRAVTCMLRKEKEAAASEIEAVSVAMEKEFELLDKAKRKTQKRGITNTLEESSSLKRKRLSDSKRKNTCGGFLGETCLSESSDGSSSEDAESSSLMNVQRRTAAKEPKTSASDSQNSVKEAPVKNGGATTSSSSDSDDDGGKEKMVLVTARSVFGKKRRKLRRA RGRKRKT

In various embodiments, the prime editor fusion proteins contemplatedherein may include any flap endonuclease variant of the above-disclosedsequences having an amino acid sequence that is at least about 70%identical, at least about 80% identical, at least about 90% identical,at least about 95% identical, at least about 96% identical, at leastabout 97% identical, at least about 98% identical, at least about 99%identical, at least about 99.5% identical, or at least about 99.9%identical to any of the above sequences.

Other endonucleases that may be utilized by the instant methods tofacilitate removal of the 5′ end single strand DNA flap include, but arenot limited to (1) trex 2, (2) exo1 endonuclease (e.g., Keijzers et al.,Biosci Rep. 2015, 35(3): e00206)

Trex 2

3′ three prime repair exonuclease 2 (TREX2) - humanAccession No. NM_080701 (SEQ ID NO: 3865) MSEAPRAETFVFLDLEATGLPSVEPEIAELSLFAVHRSSLENPEHDESGALVLPRVLDKLTLCMCPERPFTAKASEITGLSSEGLARCRKAGFDGAVVRTLQAFLSRQAGPICLVAHNGFDYDFPLLCAELRRLGARLPRDTVCLDTLPALRGLDRAHSHGTRARGRQGYSLGSLFHRYFRAEPSAAHSAEGDVHTLLLIFLHRAAELLAWADEQARGWAHIEPMYLPPDDPSLEA.3′ three prime repair exonuclease 2 (TREX2) - mouseAccession No. NM_011907 (SEQ ID NO: 3866)MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA.3′ three prime repair exonuclease 2 (TREX2) - ratAccession No. NM_001107580 (SEQ ID NO: 3867)MSEPLRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSEGLMNCRKAAFNDAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPRDTVCLDTLPALRGLDRVHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVNTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA.

ExoI

Human exonuclease 1 (EXO1) has been implicated in many different DNAmetabolic processes, including DNA mismatch repair (MMR), micro-mediatedend-joining, homologous recombination (HR), and replication. Human EXO1belongs to a family of eukaryotic nucleases, Rad2/XPG, which alsoinclude FEN1 and GEN1. The Rad2/XPG family is conserved in the nucleasedomain through species from phage to human. The EXO1 gene productexhibits both 5′ exonuclease and 5′ flap activity. Additionally, EXO1contains an intrinsic 5′ RNase H activity. Human EXO1 has a highaffinity for processing double stranded DNA (dsDNA), nicks, gaps, pseudoY structures and can resolve Holliday junctions using its inherit flapactivity. Human EXO1 is implicated in MMR and contain conserved bindingdomains interacting directly with MLH1 and MSH2. EXO1 nucleolyticactivity is positively stimulated by PCNA, MutSα (MSH2/MSH6 complex),14-3-3, MRN and 9-1-1 complex.

exonuclease 1 (EXO1) Accession No. NM_003686(Homo sapiens exonuclease 1 (EXO1), transcript variant 3) - isoform A(SEQ ID NO: 3868) MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCMKFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANLLKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRPRSAELSEDDLLSQYSLSFTKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHNAENK PGLQIKLNELWKNFGFKKF.exonuclease 1 (EXO1) Accession No. NM_006027(Homo sapiens exonuclease 1 (EXO1), transcript variant 3) - isoform B(SEQ ID NO: 3869) MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCMKFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANLLKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRPRSAELSEDDLLSQYSLSFTKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFN KPECGRVQRAIFQ.exonuclease 1 (EXO1) Accession No. NM_001319224(Homo sapiens exonuclease 1 (EXO1), transcript variant 4) - isoform C(SEQ ID NO: 3870) MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCMKFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANLLKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSIVKRPRSELSEDDLLSQYSLSFTKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKP ECGRVQRAIFQ.

D. Inteins and Split-Inteins

It will be understood that in some embodiments (e.g., delivery of aprime editor in vivo using AAV particles), it may be advantageous tosplit a polypeptide (e.g., a deaminase or a napDNAbp) or a fusionprotein (e.g., a prime editor) into an N-terminal half and a C-terminalhalf, delivery them separately, and then allow their colocalization toreform the complete protein (or fusion protein as the case may be)within the cell. Separate halves of a protein or a fusion protein mayeach comprise a split-intein tag to facilitate the reformation of thecomplete protein or fusion protein by the mechanism of protein transsplicing.

Protein trans-splicing, catalyzed by split inteins, provides an entirelyenzymatic method for protein ligation. A split-intein is essentially acontiguous intein (e.g. a mini-intein) split into two pieces namedN-intein and C-intein, respectively. The N-intein and C-intein of asplit intein can associate non-covalently to form an active intein andcatalyze the splicing reaction essentially in same way as a contiguousintein does. Split inteins have been found in nature and also engineeredin laboratories. As used herein, the term “split intein” refers to anyintein in which one or more peptide bond breaks exists between theN-terminal and C-terminal amino acid sequences such that the N-terminaland C-terminal sequences become separate molecules that cannon-covalently reassociate, or reconstitute, into an intein that isfunctional for trans-splicing reactions. Any catalytically activeintein, or fragment thereof, may be used to derive a split intein foruse in the methods of the invention. For example, in one aspect thesplit intein may be derived from a eukaryotic intein. In another aspect,the split intein may be derived from a bacterial intein. In anotheraspect, the split intein may be derived from an archaeal intein.Preferably, the split intein so-derived will possess only the amino acidsequences essential for catalyzing trans-splicing reactions.

As used herein, the “N-terminal split intein (In)” refers to any inteinsequence that comprises an N-terminal amino acid sequence that isfunctional for trans-splicing reactions. An In thus also comprises asequence that is spliced out when trans-splicing occurs. An In cancomprise a sequence that is a modification of the N-terminal portion ofa naturally occurring intein sequence. For example, an In can compriseadditional amino acid residues and/or mutated residues so long as theinclusion of such additional and/or mutated residues does not render theIn non-functional in trans-splicing. Preferably, the inclusion of theadditional and/or mutated residues improves or enhances thetrans-splicing activity of the In.

As used herein, the “C-terminal split intein (Ic)” refers to any inteinsequence that comprises a C-terminal amino acid sequence that isfunctional for trans-splicing reactions. In one aspect, the Ic comprises4 to 7 contiguous amino acid residues, at least 4 amino acids of whichare from the last β-strand of the intein from which it was derived. AnIc thus also comprises a sequence that is spliced out whentrans-splicing occurs. An Ic can comprise a sequence that is amodification of the C-terminal portion of a naturally occurring inteinsequence. For example, an Ic can comprise additional amino acid residuesand/or mutated residues so long as the inclusion of such additionaland/or mutated residues does not render the In non-functional intrans-splicing. Preferably, the inclusion of the additional and/ormutated residues improves or enhances the trans-splicing activity of theIc.

In some embodiments of the invention, a peptide linked to an Ic or an Incan comprise an additional chemical moiety including, among others,fluorescence groups, biotin, polyethylene glycol (PEG), amino acidanalogs, unnatural amino acids, phosphate groups, glycosyl groups,radioisotope labels, and pharmaceutical molecules. In other embodiments,a peptide linked to an Ic can comprise one or more chemically reactivegroups including, among others, ketone, aldehyde, Cys residues and Lysresidues. The N-intein and C-intein of a split intein can associatenon-covalently to form an active intein and catalyze the splicingreaction when an “intein-splicing polypeptide (ISP)” is present. As usedherein, “intein-splicing polypeptide (ISP)” refers to the portion of theamino acid sequence of a split intein that remains when the Ic, In, orboth, are removed from the split intein. In certain embodiments, the Incomprises the ISP. In another embodiment, the Ic comprises the ISP. Inyet another embodiment, the ISP is a separate peptide that is notcovalently linked to In nor to Ic.

Split inteins may be created from contiguous inteins by engineering oneor more split sites in the unstructured loop or intervening amino acidsequence between the −12 conserved beta-strands found in the structureof mini-inteins. Some flexibility in the position of the split sitewithin regions between the beta-strands may exist, provided thatcreation of the split will not disrupt the structure of the intein, thestructured beta-strands in particular, to a sufficient degree thatprotein splicing activity is lost.

In protein trans-splicing, one precursor protein consists of an N-exteinpart followed by the N-intein, another precursor protein consists of theC-intein followed by a C-extein part, and a trans-splicing reaction(catalyzed by the N- and C-inteins together) excises the two inteinsequences and links the two extein sequences with a peptide bond.Protein trans-splicing, being an enzymatic reaction, can work with verylow (e.g. micromolar) concentrations of proteins and can be carried outunder physiological conditions.

Exemplary sequences are as follows:

NAME SEQUENCE OF LIGAND-DEPENDENT INTEIN 2-4 INTEIN:CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH NC (SEQ ID NO: 8)3-2 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYTNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHN C (SEQ ID NO: 9)30R3-1 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV VHNC (SEQ ID NO: 10)30R3-2 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV VHNC (SEQ ID NO: 11)30R3-3 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV VHNC (SEQ ID NO: 12)37R3-1 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYNPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV VHNC ((SEQ ID NO: 13)37R3-2 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH NC (SEQ ID NO: 14)37R3-3 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH NC (SEQ ID NO: 15)

Although inteins are most frequently found as a contiguous domain, someexist in a naturally split form. In this case, the two fragments areexpressed as separate polypeptides and must associate before splicingtakes place, so-called protein trans-splicing.

An exemplary split intein is the Ssp DnaE intein, which comprises twosubunits, namely, DnaE-N and DnaE-C. The two different subunits areencoded by separate genes, namely dnaE-n and dnaE-c, which encode theDnaE-N and DnaE-C subunits, respectively. DnaE is a naturally occurringsplit intein in Synechocystis sp. PCC6803 and is capable of directingtrans-splicing of two separate proteins, each comprising a fusion witheither DnaE-N or DnaE-C.

Additional naturally occurring or engineered split-intein sequences areknown in the or can be made from whole-intein sequences described hereinor those available in the art. Examples of split-intein sequences can befound in Stevens et al., “A promiscuous split intein with expandedprotein engineering applications,” PNAS, 2017, Vol. 114: 8538-8543; Iwaiet al., “Highly efficient protein trans-splicing by a naturally splitDnaE intein from Nostoc punctiforme, FEBS Lett, 580: 1853-1858, each ofwhich are incorporated herein by reference. Additional split inteinsequences can be found, for example, in WO 2013/045632, WO 2014/055782,WO 2016/069774, and EP2877490, the contents each of which areincorporated herein by reference.

In addition, protein splicing in trans has been described in vivo and invitro (Shingledecker, et al., Gene 207:187 (1998), Southworth, et al.,EMBO J. 17:918 (1998); Mills, et al., Proc. Natl. Acad. Sci. USA,95:3543-3548 (1998); Lew, et al., J. Biol. Chem., 273:15887-15890(1998); Wu, et al., Biochim. Biophys. Acta 35732:1 (1998b), Yamazaki, etal., J. Am. Chem. Soc. 120:5591 (1998), Evans, et al., J. Biol. Chem.275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999);Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc.Natl. Acad. Sci. USA 96:13638-13643 (1999)) and provides the opportunityto express a protein as to two inactive fragments that subsequentlyundergo ligation to form a functional product, e.g., as shown in FIGS.66 and 67 with regard to the formation of a complete PE fusion proteinfrom two separately-expressed halves.

E. RNA-Protein Interaction Domain

In various embodiments, two separate protein domains (e.g., a Cas9domain and a polymerase domain) may be colocalized to one another toform a functional complex (akin to the function of a fusion proteincomprising the two separate protein domains) by using an “RNA-proteinrecruitment system,” such as the “MS2 tagging technique.” Such systemsgenerally tag one protein domain with an “RNA-protein interactiondomain” (aka “RNA-protein recruitment domain”) and the other with an“RNA-binding protein” that specifically recognizes and binds to theRNA-protein interaction domain, e.g., a specific hairpin structure.These types of systems can be leveraged to colocalize the domains of aprime editor, as well as to recruitment additional functionalities to aprime editor, such as a UGI domain. In one example, the MS2 taggingtechnique is based on the natural interaction of the MS2 bacteriophagecoat protein (“MCP” or “MS2cp”) with a stem-loop or hairpin structurepresent in the genome of the phage, i.e., the “MS2 hairpin.” In the caseof the MS2 hairpin, it is recognized and bound by the MS2 bacteriophagecoat protein (MCP). Thus, in one exemplary scenario a deaminase-MS2fusion can recruit a Cas9-MCP fusion.

A review of other modular RNA-protein interaction domains are describedin the art, for example, in Johansson et al., “RNA recognition by theMS2 phage coat protein,” Sem Virol., 1997, Vol. 8(3): 176-185;Delebecque et al., “Organization of intracellular reactions withrationally designed RNA assemblies,” Science, 2011, Vol. 333: 470-474;Mali et al., “Cas9 transcriptional activators for target specificityscreening and paired nickases for cooperative genome engineering,” Nat.Biotechnol., 2013, Vol. 31: 833-838; and Zalatan et al., “Engineeringcomplex synthetic transcriptional programs with CRISPR RNA scaffolds,”Cell, 2015, Vol. 160: 339-350, each of which are incorporated herein byreference in their entireties. Other systems include the PP7 hairpin,which specifically recruits the PCP protein, and the “com” hairpin,which specifically recruits the Com protein. See Zalatan et al.

The nucleotide sequence of the MS2 hairpin (or equivalently referred toas the “MS2 aptamer”) is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO:763).

The amino acid sequence of the MCP or MS2cp is:

(SEQ ID NO: 764) GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY.

F. UGI Domain

In other embodiments, the prime editors described herein may compriseone or more uracil glycosylase inhibitor domains. The term “uracilglycosylase inhibitor (UGI)” or “UGI domain,” as used herein, refers toa protein that is capable of inhibiting a uracil-DNA glycosylasebase-excision repair enzyme. In some embodiments, a UGI domain comprisesa wild-type UGI or a UGI as set forth in SEQ ID NO: 3873. In someembodiments, the UGI proteins provided herein include fragments of UGIand proteins homologous to a UGI or a UGI fragment. For example, in someembodiments, a UGI domain comprises a fragment of the amino acidsequence set forth in SEQ ID NO: 3873. In some embodiments, a UGIfragment comprises an amino acid sequence that comprises at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% of the amino acid sequence as set forth inSEQ ID NO: 3873. In some embodiments, a UGI comprises an amino acidsequence homologous to the amino acid sequence set forth in SEQ ID NO:3873, or an amino acid sequence homologous to a fragment of the aminoacid sequence set forth in SEQ ID NO: 3873. In some embodiments,proteins comprising UGI or fragments of UGI or homologs of UGI or UGIfragments are referred to as “UGI variants.” A UGI variant shareshomology to UGI, or a fragment thereof. For example a UGI variant is atleast 70% identical, at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, atleast 99% identical, at least 99.5% identical, or at least 99.9%identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 3873.In some embodiments, the UGI variant comprises a fragment of UGI, suchthat the fragment is at least 70% identical, at least 80% identical, atleast 90% identical, at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, at least 99% identical, atleast 99.5% identical, or at least 99.9% to the corresponding fragmentof wild-type UGI or a UGI as set forth in SEQ ID NO: 3873. In someembodiments, the UGI comprises the following amino acid sequence:

Uracil-DNA glycosylase inhibitor: >sp|P14739|UNGI_BPPB2(SEQ ID NO: 3873) MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.

The prime editors described herein may comprise more than one UGIdomain, which may be separated by one or more linkers as describedherein.

G. Additional PE Elements

In certain embodiments, the prime editors described herein may comprisean inhibitor of base repair. The term “inhibitor of base repair” or“IBR” refers to a protein that is capable in inhibiting the activity ofa nucleic acid repair enzyme, for example a base excision repair enzyme.In some embodiments, the IBR is an inhibitor of OGG base excisionrepair. In some embodiments, the IBR is an inhibitor of base excisionrepair (“iBER”). Exemplary inhibitors of base excision repair includeinhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1,hNEIL1, T7 EndoI, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, theIBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR isan iBER that may be a catalytically inactive glycosylase orcatalytically inactive dioxygenase or a small molecule or peptideinhibitor of an oxidase, or variants thereof. In some embodiments, theIBR is an iBER that may be a TDG inhibitor, MBD4 inhibitor or aninhibitor of an AlkBH enzyme. In some embodiments, the IBR is an iBERthat comprises a catalytically inactive TDG or catalytically inactiveMBD4. An exemplary catalytically inactive TDG is an N140A mutant of SEQID NO: 3872 (human TDG).

Some exemplary glycosylases are provided below. The catalyticallyinactivated variants of any of these glycosylase domains are iBERs thatmay be fused to the napDNAbp or polymerase domain of the prime editorsprovided in this disclosure.

OGG (human) (SEQ ID NO: 3937)MPARALLPRRMGHRTLASTPALWASIPCPRSELRLDLVLPSGQSFRWREQSPAHWSGVLADQVWTLTQTEEQLHCTVYRGDKSQASRPTPDELEAVRKYFQLDVTLAQLYHHWGSVDSHFQEVAQKFQGVRLLRQDPIECLFSFICSSNNNIARITGMVERLCQAFGPRLIQLDDVTYHGFPSLQALAGPEVEAHLRKLGLGYRARYVSASARAILEEQGGLAWLQQLRESSYEEAHKALCILPGVGTKVADCICLMALDKPQAVPVDVHMWHIAQRDYSWHPTTSQAKGPSPQTNKELGNFFRSLWGPYAGWAQAVLFSADLRQSRHAQEPPAKRRKGSKGPEG MPG (human)(SEQ ID NO: 3938) MVTPALQMKKPKQFCRRMGQKKQRPARAGQPHSSSDAAQAPAEQPHSSSDAAQAPCPRERCLGPPTTPGPYRSIYFSSPKGHLTRLGLEFFDQPAVPLARAFLGQVLVRRLPNGTELRGRIVETEAYLGPEDEAAHSRGGRQTPRNRGMFMKPGTLYVYIIYGMYFCMNISSQGDGACVLLRALEPLEGLETMRQLRSTLRKGTASRVLKDRELCSGPSKLCQALAINKSFDQRDLAQDEAVWLERGPLEPSEPAVVAAARVGVGHAGEWARKPLRFYVRGSPWVSVVDRVAEQDTQA MBD4 (human)(SEQ ID NO: 3871) MGTTGLESLSLGDRGAAPTVTSSERLVPDPPNDLRKEDVAMELERVGEDEEQMMIKRSSECNPLLQEPIASAQFGATAGTECRKSVPCGWERVVKQRLFGKTAGRFDVYFISPQGLKFRSKSSLANYLHKNGETSLKPEDFDFTVLSKRGIKSRYKDCSMAALTSHLQNQSNNSNWNLRTRSKCKKDVFMPPSSSSELQESRGLSNFTSTHLLLKEDEGVDDVNFRKVRKPKGKVTILKGIPIKKTKKGCRKSCSGFVQSDSKRESVCNKADAESEPVAQKSQLDRTVCISDAGACGETLSVTSEENSLVKKKERSLSSGSNFCSEQKTSGIINKFCSAKDSEHNEKYEDTFLESEEIGTKVEVVERKEHLHTDILKRGSEMDNNCSPTRKDFTGEKIFQEDTIPRTQIERRKTSLYFSSKYNKEALSPPRRKAFKKWTPPRSPFNLVQETLFHDPWKLLIATIFLNRTSGKMAIPVLWKFLEKYPSAEVARTADWRDVSELLKPLGLYDLRAKTIVKFSDEYLTKQWKYPIELHGIGKYGNDSYRIFCVNEWKQVHPEDHKLNKYHDWLWENHEKLSLS TDG (human) (SEQ ID NO: 3872)MEAENAGSYSLQQAQAFYTFPFQQLMAEAPNMAVVNEQQMPEEVPAPAPAQEPVQEAPKGRKRKPRTTEPKQPVEPKKPVESKKSGKSAKSKEKQEKITDTFKVKRKVDRFNGVSEAELLTKTLPDILTFNLDIVIIGINPGLMAAYKGHHYPGPGNHFWKCLFMSGLSEVQLNHMDDHTLPGKYGIGFTNMVERTTPGSKDLSSKEFREGGRILVQKLQKYQPRIAVFNGKCIYEIFSKEVFGVKVKNLEFGLQPHKIPDTETLCYVMPSSSARCAQFPRAQDKVHYYIKLKDLRDQLKGIERNMDVQEVQYTFDLQLAQEDAKKMAVKEEKYDPGYEAAYGGAYGENPCSSEPCGFSSNGLIESVELRGESAFSGIPNGQWMTQSFTDQIPSFSNHCG TQEQEEESHA

In some embodiments, the fusion proteins described herein may compriseone or more heterologous protein domains (e.g., about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the primeeditor components). A fusion protein may comprise any additional proteinsequence, and optionally a linker sequence between any two domains.Other exemplary features that may be present are localization sequences,such as cytoplasmic localization sequences, export sequences, such asnuclear export sequences, or other localization sequences, as well assequence tags that are useful for solubilization, purification, ordetection of the fusion proteins.

Examples of protein domains that may be fused to a prime editor orcomponent thereof (e.g., the napDNAbp domain, the polymerase domain, orthe NLS domain) include, without limitation, epitope tags, and reportergene sequences. Non-limiting examples of epitope tags include histidine(His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myctags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genesinclude, but are not limited to, glutathione-5-transferase (GST),horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT),beta-galactosidase, beta-glucuronidase, luciferase, green fluorescentprotein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), and autofluorescent proteins including bluefluorescent protein (BFP). A prime editor may be fused to a genesequence encoding a protein or a fragment of a protein that bind DNAmolecules or bind other cellular molecules, including, but not limitedto, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD)fusions, GALA DNA binding domain fusions, and herpes simplex virus (HSV)BP16 protein fusions. Additional domains that may form part of a primeeditor are described in US Patent Publication No. 2011/0059502,published Mar. 10, 2011 and incorporated herein by reference in itsentirety.

In an aspect of the disclosure, a reporter gene which includes, but isnot limited to, glutathione-5-transferase (GST), horseradish peroxidase(HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP),may be introduced into a cell to encode a gene product which serves as amarker by which to measure the alteration or modification of expressionof the gene product. In certain embodiments of the disclosure the geneproduct is luciferase. In a further embodiment of the disclosure theexpression of the gene product is decreased.

Suitable protein tags provided herein include, but are not limited to,biotin carboxylase carrier protein (BCCP) tags, myc-tags,calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags,also referred to as histidine tags or His-tags, maltose binding protein(MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, greenfluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g.,Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5tags, and SBP-tags. Additional suitable sequences will be apparent tothose of skill in the art. In some embodiments, the fusion proteincomprises one or more His tags.

In some embodiments of the present disclosure, the activity of the primeediting system may be temporally regulated by adjusting the residencetime, the amount, and/or the activity of the expressed components of thePE system. For example, as described herein, the PE may be fused with aprotein domain that is capable of modifying the intracellular half-lifeof the PE. In certain embodiments involving two or more vectors (e.g., avector system in which the components described herein are encoded ontwo or more separate vectors), the activity of the PE system may betemporally regulated by controlling the timing in which the vectors aredelivered. For example, in some embodiments a vector encoding thenuclease system may deliver the PE prior to the vector encoding thetemplate. In other embodiments, the vector encoding the PEgRNA maydeliver the guide prior to the vector encoding the PE system. In someembodiments, the vectors encoding the PE system and PEgRNA are deliveredsimultaneously. In certain embodiments, the simultaneously deliveredvectors temporally deliver, e.g., the PE, PEgRNA, and/or second strandguide RNA components. In further embodiments, the RNA (such as, e.g.,the nuclease transcript) transcribed from the coding sequence on thevectors may further comprise at least one element that is capable ofmodifying the intracellular half-life of the RNA and/or modulatingtranslational control. In some embodiments, the half-life of the RNA maybe increased. In some embodiments, the half-life of the RNA may bedecreased. In some embodiments, the element may be capable of increasingthe stability of the RNA. In some embodiments, the element may becapable of decreasing the stability of the RNA. In some embodiments, theelement may be within the 3′ UTR of the RNA. In some embodiments, theelement may include a polyadenylation signal (PA). In some embodiments,the element may include a cap, e.g., an upstream mRNA or PEgRNA end. Insome embodiments, the RNA may comprise no PA such that it is subject toquicker degradation in the cell after transcription. In someembodiments, the element may include at least one AU-rich element (ARE).The AREs may be bound by ARE binding proteins (ARE-BPs) in a manner thatis dependent upon tissue type, cell type, timing, cellular localization,and environment. In some embodiments the destabilizing element maypromote RNA decay, affect RNA stability, or activate translation. Insome embodiments, the ARE may comprise 50 to 150 nucleotides in length.In some embodiments, the ARE may comprise at least one copy of thesequence AUUUA. In some embodiments, at least one ARE may be added tothe 3′ UTR of the RNA. In some embodiments, the element may be aWoodchuck Hepatitis Virus (WHP).

Posttranscriptional Regulatory Element (WPRE), which creates a tertiarystructure to enhance expression from the transcript. In furtherembodiments, the element is a modified and/or truncated WPRE sequencethat is capable of enhancing expression from the transcript, asdescribed, for example in Zufferey et al., J Virol, 73(4): 2886-92(1999) and Flajolet et al., J Virol, 72(7): 6175-80 (1998). In someembodiments, the WPRE or equivalent may be added to the 3′ UTR of theRNA. In some embodiments, the element may be selected from other RNAsequence motifs that are enriched in either fast- or slow-decayingtranscripts.

In some embodiments, the vector encoding the PE or the PEgRNA may beself-destroyed via cleavage of a target sequence present on the vectorby the PE system. The cleavage may prevent continued transcription of aPE or a PEgRNA from the vector. Although transcription may occur on thelinearized vector for some amount of time, the expressed transcripts orproteins subject to intracellular degradation will have less time toproduce off-target effects without continued supply from expression ofthe encoding vectors.

[6] PEgRNAs

The prime editing system described herein contemplates the use of anysuitable PEgRNAs. The inventors have discovered that the mechanism oftarget-primed reverse transcription (TPRT) can be leveraged or adaptedfor conducting precision and versatile CRISPR/Cas-based genome editingthrough the use of a specially configured guide RNA comprising a reversetranscription (RT) template sequence that codes for the desirednucleotide change. The application refers to this specially configuredguide RNA as an “extended guide RNA” or a “PEgRNA” since the RT templatesequence can be provided as an extension of a standard or traditionalguide RNA molecule. The application contemplates any suitableconfiguration or arrangement for the extended guide RNA.

PEgRNA Architecture

FIG. 3A shows one embodiment of an extended guide RNA usable in theprime editing system disclosed herein whereby a traditional guide RNA(the dotted portion) includes a ˜20 nt protospacer sequence and a gRNAcore region, which binds with the napDNAbp. In this embodiment, theguide RNA includes an extended RNA segment at the 5′ end, i.e., a 5′extension. In this embodiment, the 5′extension includes a reversetranscription template sequence, a reverse transcription primer bindingsite, and an optional 5-20 nucleotide linker sequence. As shown in FIGS.1A-1B, the RT primer binding site hybrizes to the free 3′ end that isformed after a nick is formed in the non-target strand of the R-loop,thereby priming reverse transcriptase for DNA polymerization in the5′-3′ direction.

FIG. 3B shows another embodiment of an extended guide RNA usable in theprime editing system disclosed herein whereby a traditional guide RNA(the dotted portion) includes a ˜20 nt protospacer sequence and a gRNAcore, which binds with the napDNAbp. In this embodiment, the guide RNAincludes an extended RNA segment at the 3′ end, i.e., a 3′ extension. Inthis embodiment, the 3′extension includes a reverse transcriptiontemplate sequence, and a reverse transcription primer binding site. Asshown in FIGS. 1C-1D, the RT primer binding site hybrizes to the free 3′end that is formed after a nick is formed in the non-target strand ofthe R-loop, thereby priming reverse transcriptase for DNA polymerizationin the 5′-3′ direction.

FIG. 3C shows another embodiment of an extend guide RNA usable in theprime editing system disclosed herein whereby a traditional guide RNA(the dotted portion) includes a ˜20 nt protospacer sequence and a gRNAcore, which binds with the napDNAbp. In this embodiment, the guide RNAincludes an extended RNA segment at an intermolecular position withinthe gRNA core, i.e., an intramolecular extension. In this embodiment,the intramolecular extension includes a reverse transcription templatesequence, and a reverse transcription primer binding site. The RT primerbinding site hybrizes to the free 3′ end that is formed after a nick isformed in the non-target strand of the R-loop, thereby priming reversetranscriptase for DNA polymerization in the 5′-3′ direction.

In one embodiment, the position of the intermolecular RNA extension isnot in the protospacer sequence of the guide RNA. In another embodiment,the position of the intermolecular RNA extension in the gRNA core. Instill another embodiment, the position of the intermolecular RNAextension is any with the guide RNA molecule except within theprotospacer sequence, or at a position which disrupts the protospacersequence.

In one embodiment, the intermolecular RNA extension is inserteddownstream from the 3′ end of the protospacer sequence. In anotherembodiment, the intermolecular RNA extension is inserted at least 1nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, atleast 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides,at least 19 nucleotides, at least 20 nucleotides, at least 21nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least24 nucleotides, at least 25 nucleotides downstream of the 3′ end of theprotospacer sequence.

In other embodiments, the intermolecular RNA extension is inserted intothe gRNA, which refers to the portion of the guide RNA corresponding orcomprising the tracrRNA, which binds and/or interacts with the Cas9protein or equivalent thereof (i.e, a different napDNAbp). Preferablythe insertion of the intermolecular RNA extension does not disrupt orminimally disrupts the interaction between the tracrRNA portion and thenapDNAbp.

The length of the RNA extension can be any useful length. In variousembodiments, the RNA extension is at least 5 nucleotides, at least 6nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, atleast 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides,at least 18 nucleotides, at least 19 nucleotides, at least 20nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, atleast 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides,at least 60 nucleotides, at least 70 nucleotides, at least 80nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, orat least 500 nucleotides in length.

The RT template sequence can also be any suitable length. For example,the RT template sequence can be at least 3 nucleotides, at least 4nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, atleast 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides,at least 19 nucleotides, at least 20 nucleotides, at least 30nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, atleast 90 nucleotides, at least 100 nucleotides, at least 200nucleotides, at least 300 nucleotides, at least 400 nucleotides, or atleast 500 nucleotides in length.

In still other embodiments, wherein the reverse transcription primerbinding site sequence is at least 3 nucleotides, at least 4 nucleotides,at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides,at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides,at least 11 nucleotides, at least 12 nucleotides, at least 13nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, atleast 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides,at least 40 nucleotides, at least 50 nucleotides, at least 60nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, or at least 500nucleotides in length.

In other embodiments, the optional linker or spacer sequence is at least3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, atleast 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides,at least 15 nucleotides, at least 16 nucleotides, at least 17nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, atleast 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides,at least 80 nucleotides, at least 90 nucleotides, at least 100nucleotides, at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, or at least 500 nucleotides in length.

The RT template sequence, in certain embodiments, encodes asingle-stranded DNA molecule which is homologous to the non-targetstrand (and thus, complementary to the corresponding site of the targetstrand) but includes one or more nucleotide changes. The least onenucleotide change may include one or more single-base nucleotidechanges, one or more deletions, and one or more insertions.

As depicted in FIG. 1G, the synthesized single-stranded DNA product ofthe RT template sequence is homologous to the non-target strand andcontains one or more nucleotide changes. The single-stranded DNA productof the RT template sequence hybridizes in equilibrium with thecomplementary target strand sequence, thereby displacing the homologousendogenous target strand sequence. The displaced endogenous strand maybe referred to in some embodiments as a 5′ endogenous DNA flap species(e.g., see FIG. 1E). This 5′ endogenous DNA flap species can be removedby a 5′ flap endonuclease (e.g., FEN1) and the single-stranded DNAproduct, now hybridized to the endogenous target strand, may be ligated,thereby creating a mismatch between the endogenous sequence and thenewly synthesized strand. The mismatch may be resolved by the cell'sinnate DNA repair and/or replication processes.

In various embodiments, the nucleotide sequence of the RT templatesequence corresponds to the nucleotide sequence of the non-target strandwhich becomes displaced as the 5′ flap species and which overlaps withthe site to be edited.

In various embodiments of the extended guide RNAs, the reversetranscription template sequence may encode a single-strand DNA flap thatis complementary to an endogenous DNA sequence adjacent to a nick site,wherein the single-strand DNA flap comprises a desired nucleotidechange. The single-stranded DNA flap may displace an endogenoussingle-strand DNA at the nick site. The displaced endogenoussingle-strand DNA at the nick site can have a 5′ end and form anendogenous flap, which can be excised by the cell. In variousembodiments, excision of the 5′ end endogenous flap can help driveproduct formation since removing the 5′ end endogenous flap encourageshybridization of the single-strand 3′ DNA flap to the correspondingcomplementary DNA strand, and the incorporation or assimilation of thedesired nucleotide change carried by the single-strand 3′ DNA flap intothe target DNA.

In various embodiments of the extended guide RNAs, the cellular repairof the single-strand DNA flap results in installation of the desirednucleotide change, thereby forming a desired product.

In still other embodiments, the desired nucleotide change is installedin an editing window that is between about −5 to +5 of the nick site, orbetween about −10 to +10 of the nick site, or between about −20 to +20of the nick site, or between about −30 to +30 of the nick site, orbetween about −40 to +40 of the nick site, or between about −50 to +50of the nick site, or between about −60 to +60 of the nick site, orbetween about −70 to +70 of the nick site, or between about −80 to +80of the nick site, or between about −90 to +90 of the nick site, orbetween about −100 to +100 of the nick site, or between about −200 to+200 of the nick site.

In other embodiments, the desired nucleotide change is installed in anediting window that is between about +1 to +2 from the nick site, orabout +1 to +3, +1 to +4, +1 to +5, +1 to +6, +1 to +7, +1 to +8, +1 to+9, +1 to +10, +1 to +11, +1 to +12, +1 to +13, +1 to +14, +1 to +15, +1to +16, +1 to +17, +1 to +18, +1 to +19, +1 to +20, +1 to +21, +1 to+22, +1 to +23, +1 to +24, +1 to +25, +1 to +26, +1 to +27, +1 to +28,+1 to +29, +1 to +30, +1 to +31, +1 to +32, +1 to +33, +1 to +34, +1 to+35, +1 to +36, +1 to +37, +1 to +38, +1 to +39, +1 to +40, +1 to +41,+1 to +42, +1 to +43, +1 to +44, +1 to +45, +1 to +46, +1 to +47, +1 to+48, +1 to +49, +1 to +50, +1 to +51, +1 to +52, +1 to +53, +1 to +54,+1 to +55, +1 to +56, +1 to +57, +1 to +58, +1 to +59, +1 to +60, +1 to+61, +1 to +62, +1 to +63, +1 to +64, +1 to +65, +1 to +66, +1 to +67,+1 to +68, +1 to +69, +1 to +70, +1 to +71, +1 to +72, +1 to +73, +1 to+74, +1 to +75, +1 to +76, +1 to +77, +1 to +78, +1 to +79, +1 to +80,+1 to +81, +1 to +82, +1 to +83, +1 to +84, +1 to +85, +1 to +86, +1 to+87, +1 to +88, +1 to +89, +1 to +90, +1 to +90, +1 to +91, +1 to +92,+1 to +93, +1 to +94, +1 to +95, +1 to +96, +1 to +97, +1 to +98, +1 to+99, +1 to +100, +1 to +101, +1 to +102, +1 to +103, +1 to +104, +1 to+105, +1 to +106, +1 to +107, +1 to +108, +1 to +109, +1 to +110, +1 to+111, +1 to +112, +1 to +113, +1 to +114, +1 to +115, +1 to +116, +1 to+117, +1 to +118, +1 to +119, +1 to +120, +1 to +121, +1 to +122, +1 to+123, +1 to +124, or +1 to +125 from the nick site.

In still other embodiments, the desired nucleotide change is installedin an editing window that is between about +1 to +2 from the nick site,or about +1 to +5, +1 to +10, +1 to +15, +1 to +20, +1 to +25, +1 to+30, +1 to +35, +1 to +40, +1 to +45, +1 to +50, +1 to +55, +1 to +100,+1 to +105, +1 to +110, +1 to +115, +1 to +120, +1 to +125, +1 to +130,+1 to +135, +1 to +140, +1 to +145, +1 to +150, +1 to +155, +1 to +160,+1 to +165, +1 to +170, +1 to +175, +1 to +180, +1 to +185, +1 to +190,+1 to +195, or +1 to +200, from the nick site.

In various aspects, the extended guide RNAs are modified versions of aguide RNA. Guide RNAs maybe naturally occurring, expressed from anencoding nucleic acid, or synthesized chemically. Methods are well knownin the art for obtaining or otherwise synthesizing guide RNAs and fordetermining the appropriate sequence of the guide RNA, including theprotospacer sequence which interacts and hybridizes with the targetstrand of a genomic target site of interest.

In various embodiments, the particular design aspects of a guide RNAsequence will depend upon the nucleotide sequence of a genomic targetsite of interest (i.e., the desired site to be edited) and the type ofnapDNAbp (e.g., Cas9 protein) present in prime editing systems describedherein, among other factors, such as PAM sequence locations, percent G/Ccontent in the target sequence, the degree of microhomology regions,secondary structures, etc.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a napDNAbp (e.g., a Cas9, Cas9 homolog, or Cas9 variant) to thetarget sequence. In some embodiments, the degree of complementaritybetween a guide sequence and its corresponding target sequence, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences, non-limiting example of which includethe Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies,ELAND (Illumina, San Diego, Calif.), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.

In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of aguide sequence to direct sequence-specific binding of a prime editor(PE) to a target sequence may be assessed by any suitable assay. Forexample, the components of a prime editor (PE), including the guidesequence to be tested, may be provided to a host cell having thecorresponding target sequence, such as by transfection with vectorsencoding the components of a prime editor (PE) disclosed herein,followed by an assessment of preferential cleavage within the targetsequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target polynucleotide sequence may be evaluated in a testtube by providing the target sequence, components of a prime editor(PE), including the guide sequence to be tested and a control guidesequence different from the test guide sequence, and comparing bindingor rate of cleavage at the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome. For example, for the S. pyogenes Cas9, a unique targetsequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 204) where NNNNNNNNNNNNXGG (SEQ IDNO: 205) (N is A, G, T, or C; and X can be anything). A unique targetsequence in a genome may include an S. pyogenes Cas9 target site of theform MMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 206) where NNNNNNNNNNNXGG (SEQID NO: 207) (N is A, G, T, or C; and X can be anything). For the S.thermophilus CRISPR1Cas9, a unique target sequence in a genome mayinclude a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQID NO: 208) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 209) (N is A, G, T, orC; X can be anything; and W is A or T). A unique target sequence in agenome may include an S. thermophilus CRISPR 1 Cas9 target site of theform MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 210) whereNNNNNNNNNNNXXAGAAW (SEQ ID NO: 211) (N is A, G, T, or C; X can beanything; and W is A or T). For the S. pyogenes Cas9, a unique targetsequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGGXG (SEQ ID NO: 212) where NNNNNNNNNNNNXGGXG (SEQID NO: 213) (N is A, G, T, or C; and X can be anything). A unique targetsequence in a genome may include an S. pyogenes Cas9 target site of theform MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 214) where NNNNNNNNNNNXGGXG(SEQ ID NO: 215) (N is A, G, T, or C; and X can be anything). In each ofthese sequences “M” may be A, G, T, or C, and need not be considered inidentifying a sequence as unique.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and P A Carr and G M Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSer. No. 61/836,080; Broad Reference BI-2013/004A); incorporated hereinby reference.

In general, a tracr mate sequence includes any sequence that hassufficient complementarity with a tracr sequence to promote one or moreof: (1) excision of a guide sequence flanked by tracr mate sequences ina cell containing the corresponding tracr sequence; and (2) formation ofa complex at a target sequence, wherein the complex comprises the tracrmate sequence hybridized to the tracr sequence. In general, degree ofcomplementarity is with reference to the optimal alignment of the tracrmate sequence and tracr sequence, along the length of the shorter of thetwo sequences. Optimal alignment may be determined by any suitablealignment algorithm, and may further account for secondary structures,such as self-complementarity within either the tracr sequence or tracrmate sequence. In some embodiments, the degree of complementaritybetween the tracr sequence and tracr mate sequence along the length ofthe shorter of the two when optimally aligned is about or more thanabout 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, orhigher. In some embodiments, the tracr sequence is about or more thanabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,40, 50, or more nucleotides in length. In some embodiments, the tracrsequence and tracr mate sequence are contained within a singletranscript, such that hybridization between the two produces atranscript having a secondary structure, such as a hairpin. Preferredloop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG. In anembodiment of the invention, the transcript or transcribedpolynucleotide sequence has at least two or more hairpins. In preferredembodiments, the transcript has two, three, four or five hairpins. In afurther embodiment of the invention, the transcript has at most fivehairpins. In some embodiments, the single transcript further includes atranscription termination sequence; preferably this is a polyT sequence,for example six T nucleotides. Further non-limiting examples of singlepolynucleotides comprising a guide sequence, a tracr mate sequence, anda tracr sequence are as follows (listed 5′ to 3′), where “N” representsa base of a guide sequence, the first block of lower case lettersrepresent the tracr mate sequence, and the second block of lower caseletters represent the tracr sequence, and the final poly-T sequencerepresents the transcription terminator:

(1) (SEQ ID NO: 216) NNNNNNNNGTTTTTGTACTCTCAAGATTTAGAAATAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAATTTTTT; (2) (SEQ ID NO: 217)NNNNNNNNNNNNNNNNNNGTTTTTGTACTCTCAGAAATGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAATTTTTT; (3) (SEQ ID NO: 218)NNNNNNNNNNNNNNNNNNNNGTTTTTGTACTCTCAGAAATGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGG GTGTTTTTT; (4)(SEQ ID NO: 219) NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TT; (5) (SEQ ID NO: 220) NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGTTTTTTT; AND (6) (SEQ ID NO: 221)NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCATTTTTTTT.

In some embodiments, sequences (1) to (3) are used in combination withCas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to(6) are used in combination with Cas9 from S. pyogenes. In someembodiments, the tracr sequence is a separate transcript from atranscript comprising the tracr mate sequence.

It will be apparent to those of skill in the art that in order to targetany of the fusion proteins comprising a Cas9 domain and asingle-stranded DNA binding protein, as disclosed herein, to a targetsite, e.g., a site comprising a point mutation to be edited, it istypically necessary to co-express the fusion protein together with aguide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein,a guide RNA typically comprises a tracrRNA framework allowing for Cas9binding, and a guide sequence, which confers sequence specificity to theCas9:nucleic acid editing enzyme/domain fusion protein.

In some embodiments, the guide RNA comprises a structure 5′-[guidesequence]-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU-3′ (SEQ ID NO: 222), wherein the guidesequence comprises a sequence that is complementary to the targetsequence. The guide sequence is typically 20 nucleotides long. Thesequences of suitable guide RNAs for targeting Cas9:nucleic acid editingenzyme/domain fusion proteins to specific genomic target sites will beapparent to those of skill in the art based on the instant disclosure.Such suitable guide RNA sequences typically comprise guide sequencesthat are complementary to a nucleic sequence within 50 nucleotidesupstream or downstream of the target nucleotide to be edited. Someexemplary guide RNA sequences suitable for targeting any of the providedfusion proteins to specific target sequences are provided herein.Additional guide sequences are well known in the art and can be usedwith the prime editor (PE) described herein.

In other embodiments, the PEgRNAs include those depicted in FIG. 3D.

In still other embodiments, the PEgRNAs may include those depicted inFIG. 3E.

FIG. 3D provides the structure of an embodiment of a PEgRNA contemplatedherein and which may be designed in accordance with the methodologydefined in Example 2. The PEgRNA comprises three main component elementsordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and anextension arm at the 3′ end. The extension arm may further be dividedinto the following structural elements in the 5′ to 3′ direction,namely: a primer binding site (A), an edit template (B), and a homologyarm (C). In addition, the PEgRNA may comprise an optional 3′ endmodifier region (e1) and an optional 5′ end modifier region (e2). Stillfurther, the PEgRNA may comprise a transcriptional termination signal atthe 3′ end of the PEgRNA (not depicted). These structural elements arefurther defined herein. The depiction of the structure of the PEgRNA isnot meant to be limiting and embraces variations in the arrangement ofthe elements. For example, the optional sequence modifiers (e1) and (e2)could be positioned within or between any of the other regions shown,and not limited to being located at the 3′ and 5′ ends.

FIG. 3E provides the structure of another embodiment of a PEgRNAcontemplated herein and which may be designed in accordance with themethodology defined in Example 2. The PEgRNA comprises three maincomponent elements ordered in the 5′ to 3′ direction, namely: a spacer,a gRNA core, and an extension arm at the 3′ end. The extension arm mayfurther be divided into the following structural elements in the 5′ to3′ direction, namely: a primer binding site (A), an edit template (B),and a homology arm (C). In addition, the PEgRNA may comprise an optional3′ end modifier region (e1) and an optional 5′ end modifier region (e2).Still further, the PEgRNA may comprise a transcriptional terminationsignal on the 3′ end of the PEgRNA (not depicted). These structuralelements are further defined herein. The depiction of the structure ofthe PEgRNA is not meant to be limiting and embraces variations in thearrangement of the elements. For example, the optional sequencemodifiers (e1) and (e2) could be positioned within or between any of theother regions shown, and not limited to being located at the 3′ and 5′ends.

PEgRNA Improvements

The PEgRNAs may also include additional design improvements that maymodify the properties and/or characteristics of PEgRNAs therebyimproving the efficacy of prime editing. In various embodiments, theseimprovements may belong to one or more of a number of differentcategories, including but not limited to: (1) designs to enableefficient expression of functional PEgRNAs from non-polymerase III (polIII) promoters, which would enable the expression of longer PEgRNAswithout burdensome sequence requirements; (2) improvements to the core,Cas9-binding PEgRNA scaffold, which could improve efficacy; (3)modifications to the PEgRNA to improve RT processivity, enabling theinsertion of longer sequences at targeted genomic loci; and (4) additionof RNA motifs to the 5′ or 3′ termini of the PEgRNA that improve PEgRNAstability, enhance RT processivity, prevent misfolding of the PEgRNA, orrecruit additional factors important for genome editing.

In one embodiment, PEgRNA could be designed with polII promoters toimprove the expression of longer-length PEgRNA with larger extensionarms. sgRNAs are typically expressed from the U6 snRNA promoter. Thispromoter recruits pol III to express the associated RNA and is usefulfor expression of short RNAs that are retained within the nucleus.However, pol III is not highly processive and is unable to express RNAslonger than a few hundred nucleotides in length at the levels requiredfor efficient genome editing. Additionally, pol III can stall orterminate at stretches of U's, potentially limiting the sequencediversity that could be inserted using a PEgRNA. Other promoters thatrecruit polymerase II (such as pCMV) or polymerase I (such as the U1snRNA promoter) have been examined for their ability to express longersgRNAs. However, these promoters are typically partially transcribed,which would result in extra sequence 5′ of the spacer in the expressedPEgRNA, which has been shown to result in markedly reduced Cas9:sgRNAactivity in a site-dependent manner. Additionally, while polII-transcribed PEgRNAs can simply terminate in a run of 6-7 U's, PEgRNAstranscribed from pol II or pol I would require a different terminationsignal. Often such signals also result in polyadenylation, which wouldresult in undesired transport of the PEgRNA from the nucleus. Similarly,RNAs expressed from pol II promoters such as pCMV are typically5′-capped, also resulting in their nuclear export.

Previously, Rinn and coworkers screened a variety of expressionplatforms for the production of long-noncoding RNA-(lncRNA) taggedsgRNAs¹⁸³. These platforms include RNAs expressed from pCMV and thatterminate in the ENE element from the MALAT1 ncRNA from humans¹⁸⁴, thePAN ENE element from KSHV¹⁸⁵, or the 3′ box from U1 snRNA^(184, 187).Notably, the MALAT1 ncRNA and PAN ENEs form triple helices protectingthe polyA-tail^(184,187). These constructs could also enhance RNAstability. It is contemplated that these expression systems will alsoenable the expression of longer PEgRNAs.

In addition, a series of methods have been designed for the cleavage ofthe portion of the pol II promoter that would be transcribed as part ofthe PEgRNA, adding either a self-cleaving ribozyme such as thehammerhead¹⁸⁸, pistol¹⁸⁹, hatchet¹⁸⁹, hairpin¹⁹⁰, VS¹⁹¹, twister¹⁹², ortwister sister¹⁹² ribozymes, or other self-cleaving elements to processthe transcribed guide, or a hairpin that is recognized by Csy4¹⁹³ andalso leads to processing of the guide. Also, it is hypothesized thatincorporation of multiple ENE motifs could lead to improved PEgRNAexpression and stability, as previously demonstrated for the KSHV PANRNA and element¹⁸⁵. It is also anticipated that circularizing the PEgRNAin the form of a circular intronic RNA (ciRNA) could also lead toenhanced RNA expression and stability, as well as nuclearlocalization¹⁹⁴.

In various embodiments, the PEgRNA may include various above elements,as exemplified by the following sequence.

Non-limiting example 1 - PEgRNA expression platform consistingof pCMV, Csy4 hairpin, the PEgRNA, and MALAT1 ENE (SEQ ID NO: 223)TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGATGCTGGTGGTTGGCACTCCTGGTTTCCAGGACGGGGTTCAAATCCCTGCGGCGTCT TTGCTTTGACTNon-limiting example 2-PEgRNA expression platform consistingof pCMV, Csy4 hairing, the PEgRNA, and PAN ENE (SEQ ID NO: 224)TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAANon-limiting example 3 - PEgRNA expression platform consistingof pCMV, Csy4 hairing, the PEgRNA, and 3xPAN ENE (SEQ ID NO: 225)TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAAACACACTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAATCTCTCTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAANon-limiting example 4 - PEgRNA expression platform consistingof pCMV, Csy4 hairing, the PEgRNA, and 3′box (SEQ ID NO: 226)TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAANon-limiting example 5 - PEgRNA expression platform consistingof pU1, Csy4 hairping, the PEgRNA, and 3′box (SEQ ID NO: 227)CTAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGGAAAAAGGGAGAGGCAGACGTCACTTCCCCTTGGCGGCTCTGGCAGCAGATTGGTCGGTTGAGTGGCAGAAAGGCAGACGGGGACTGGGCAAGGCACTGTCGGTGACATCACGGACAGGGCGACTTCTATGTAGATGAGGCAGCGCAGAGGCTGCTGCTTCGCCACTTGCTGCTTCACCACGAAGGAGTTCCCGTGCCCTGGGAGCGGGTTCAGGACCGCTGATCGGAAGTGAGAATCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCGGGAGTGCGCGGGGCAAGTGACCGTGTGTGTAAAGAGTGAGGCGTATGAGGCTGTGTCGGGGCAGAGGCCCAAGATCTCAGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTCAGCAAGTTCAGAGAAATCTGAACTTGCTGGATTTTTGGAGCAGGGAGATGGAATAGGAGCTTGCTCCGTCCACTCCACGCATCGACCTGGTATTGCAGTACCTCCAGGAACGGTGCACCCACTTTCTGGAGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAA.

In various other embodiments, the PEgRNA may be improved by introducingimprovements to the scaffold or core sequences. This can be done byintroducing known

The core, Cas9-binding PEgRNA scaffold can likely be improved to enhancePE activity. Several such approaches have already been demonstrated. Forinstance, the first pairing element of the scaffold (P1) contains aGTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such runs of Ts have beenshown to result in pol III pausing and premature termination of the RNAtranscript. Rational mutation of one of the T-A pairs to a G-C pair inthis portion of P1 has been shown to enhance sgRNA activity, suggestingthis approach would also be feasible for PEgRNAs¹⁹⁵. Additionally,increasing the length of P1 has also been shown to enhance sgRNA foldingand lead to improved activity¹⁹⁵, suggesting it as another avenue forthe improvement of PEgRNA activity. Example improvements to the core caninclude:

PEgRNA containing a 6 nt extension to P1 (SEQ ID NO: 228)GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTTPEgRNA containing a T-A to G-C mutation within P1 (SEQ ID NO: 229)GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT

In various other embodiments, the PEgRNA may be improved by introducingmodifications to the edit template region. As the size of the insertiontemplated by the PEgRNA increases, it is more likely to be degraded byendonucleases, undergo spontaneous hydrolysis, or fold into secondarystructures unable to be reverse-transcribed by the RT or that disruptfolding of the PEgRNA scaffold and subsequent Cas9-RT binding.Accordingly, it is likely that modification to the template of thePEgRNA might be necessary to affect large insertions, such as theinsertion of whole genes. Some strategies to do so include theincorporation of modified nucleotides within a synthetic orsemi-synthetic PEgRNA that render the RNA more resistant to degradationor hydrolysis or less likely to adopt inhibitory secondarystructures¹⁹⁶. Such modifications could include 8-aza-7-deazaguanosine,which would reduce RNA secondary structure in G-rich sequences;locked-nucleic acids (LNA) that reduce degradation and enhance certainkinds of RNA secondary structure; 2′-O-methyl, 2′-fluoro, or2′-O-methoxyethoxy modifications that enhance RNA stability. Suchmodifications could also be included elsewhere in the PEgRNA to enhancestability and activity. Alternatively or additionally, the template ofthe PEgRNA could be designed such that it both encodes for a desiredprotein product and is also more likely to adopt simple secondarystructures that are able to be unfolded by the RT. Such simplestructures would act as a thermodynamic sink, making it less likely thatmore complicated structures that would prevent reverse transcriptionwould occur. Finally, one could also split the template into two,separate PEgRNAs. In such a design, a PE would be used to initiatetranscription and also recruit a separate template RNA to the targetedsite via an RNA-binding protein fused to Cas9 or an RNA recognitionelement on the PEgRNA itself such as the MS2 aptamer. The RT couldeither directly bind to this separate template RNA, or initiate reversetranscription on the original PEgRNA before swapping to the secondtemplate. Such an approach could enable long insertions by bothpreventing misfolding of the PEgRNA upon addition of the long templateand also by not requiring dissociation of Cas9 from the genome for longinsertions to occur, which could possibly be inhibiting PE-based longinsertions.

In still other embodiments, the PEgRNA may be improved by introducingadditional RNA motifs at the 5′ and 3′ termini of the PEgRNAs, or evenat positions therein between (e.g., in the gRNA core region, or thespacer). Several such motifs—such as the PAN ENE from KSHV and the ENEfrom MALAT1 were discussed above as possible means to terminateexpression of longer PEgRNAs from non-pol III promoters. These elementsform RNA triple helices that engulf the polyA tail, resulting in theirbeing retained within the nucleus^(184, 187) However, by forming complexstructures at the 3′ terminus of the PEgRNA that occlude the terminalnucleotide, these structures would also likely help preventexonuclease-mediated degradation of PEgRNAs.

Other structural elements inserted at the 3′ terminus could also enhanceRNA stability, albeit without enabling termination from non-pol IIIpromoters. Such motifs could include hairpins or RNA quadruplexes thatwould occlude the 3′ terminus¹⁹⁷, or self-cleaving ribozymes such as HDVthat would result in the formation of a 2′-3′-cyclic phosphate at the 3′terminus and also potentially render the PEgRNA less likely to bedegraded by exonucleases¹⁹⁸. Inducing the PEgRNA to cyclize viaincomplete splicing—to form a ciRNA—could also increase PEgRNA stabilityand result in the PEgRNA being retained within the nucleus¹⁹⁴.

Additional RNA motifs could also improve RT processivity or enhancePEgRNA activity by enhancing RT binding to the DNA-RNA duplex. Additionof the native sequence bound by the RT in its cognate retroviral genomecould enhance RT activity¹⁹⁹. This could include the native primerbinding site (PBS), polypurine tract (PPT), or kissing loops involved inretroviral genome dimerization and initiation of transcription¹⁹⁹.

Addition of dimerization motifs—such as kissing loops or a GNRAtetraloop/tetraloop receptor pair²⁰⁰—at the 5′ and 3′ termini of thePEgRNA could also result in effective circularization of the PEgRNA,improving stability. Additionally, it is envisioned that addition ofthese motifs could enable the physical separation of the PEgRNA spacerand primer, prevention occlusion of the spacer which would hinder PEactivity. Short 5′ extensions or 3′ extensions to the PEgRNA that form asmall toehold hairpin in the spacer region or along the primer bindingsite could also compete favorably against the annealing ofintracomplementary regions along the length of the PEgRNA, e.g., theinteraction between the spacer and the primer binding site that canoccur. Finally, kissing loops could also be used to recruit othertemplate RNAs to the genomic site and enable swapping of RT activityfrom one RNA to the other. As exemplary embodiments of various secondarystructures, the PEgRNA depicted in FIG. 3D and FIG. 3E list a numbersecondary RNA structures that may be engineered into any region of thePEgRNA, including in the terminal portions of the extension arm (i.e.,e1 and e2), as shown.

Example improvements include, but are not limited to:

PEgRNA-HDV fusion (SEQ ID NO: 230)GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT PEgRNA-MMLV kissing loop(SEQ ID NO: 231) GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGA GACGTCCCACCTTTTTTTPEgRNA-VS ribozyme kissing loop (SEQ ID NO: 232)GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTCCATCAGTTGACACCCTGAGGTTTTTTT PEgRNA-GNRA tetraloop/tetraloop receptor(SEQ ID NO: 233) GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTUACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTTPEgRNA template switching secondary RNA-HDV fusion (SEQ ID NO: 234)TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT

PEgRNA scaffold could be further improved via directed evolution, in ananalogous fashion to how SpCas9 and prime editor (PE) have beenimproved. Directed evolution could enhance PEgRNA recognition by Cas9 orevolved Cas9 variants. Additionally, it is likely that different PEgRNAscaffold sequences would be optimal at different genomic loci, eitherenhancing PE activity at the site in question, reducing off-targetactivities, or both. Finally, evolution of PEgRNA scaffolds to whichother RNA motifs have been added would almost certainly improve theactivity of the fused PEgRNA relative to the unevolved, fusion RNA. Forinstance, evolution of allosteric ribozymes composed of c-di-GMP-Iaptamers and hammerhead ribozymes led to dramatically improvedactivity²⁰², suggesting that evolution would improve the activity ofhammerhead-PEgRNA fusions as well. In addition, while Cas9 currentlydoes not generally tolerate 5′ extension of the sgRNA, directedevolution will likely generate enabling mutations that mitigate thisintolerance, allowing additional RNA motifs to be utilized.

The present disclosure contemplates any such ways to further improve theefficacy of the prime editing systems disclosed here.

In various embodiments, it may be advantageous to limit the appearanceof consecutive sequence of Ts from the extension arm as consecutiveseries of T's may limit the capacity of the PEgRNA to be transcribed.For example, strings of at least consecutive three T's, at leastconsecutive four T's, at least consecutive five T's, at leastconsecutive six T's, at least consecutive seven T's, at leastconsecutive eight T's, at least consecutive nine T's, at leastconsecutive ten T's, at least consecutive eleven T's, at leastconsecutive twelve T's, at least consecutive thirteen T's, at leastconsecutive fourteen T's, or at least consecutive fifteen T's should beavoided when designing the PEgRNA, or should be at least removed fromthe final designed sequence. In one embodiment, one can avoid theincludes of unwanted strings of consecutive T's in PEgRNA extension armsbut avoiding target sites that are rich in consecutive A:T nucleobasepairs.

Split PEgRNA Designs for Trans Prime Editing

The instant disclosure also contemplates trans prime editing, whichrefers to a modified version of prime editing which operates byseparating the PEgRNA into two distinct molecules: a guide RNA and atPERT molecule. The tPERT molecule is programmed to co-localize with theprime editor complex at a target DNA site, bringing the primer bindingsite and the DNA synthesis template to the prime editor in trans. Forexample, see FIG. 3G for an embodiment of a trans prime editor (tPE)which shows a two-component system comprising (1) an recruiting protein(RP)-PE:gRNA complex and (2) a tPERT that includes a primer binding siteand a DNA synthesis template joined to an RNA-protein recruitment domain(e.g., stem loop or hairpin), wherein the recruiting protein componentof the RP-PE:gRNA complex recruits the tPERT to a target site to beedited, thereby associating the PBS and DNA synthesis template with theprime editor in trans. Said another way, the tPERT is engineered tocontain (all or part of) the extension arm of a PEgRNA, which includesthe primer binding site and the DNA synthesis template. One advantage ofthis approach is to separate the extension arm of a PEgRNA from theguide RNA, thereby minimizing annealing interactions that tend to occurbetween the PBS of the extension arm and the spacer sequence of theguide RNA.

A key feature of trans prime editing is the ability of the trans primeeditor to recruit the tPERT to the site of DNA editing, therebyeffectively co-localizing all of the functions of a PEgRNA at the siteof prime editing. Recruitment can be achieve by installing anRNA-protein recruitment domain, such as a MS2 aptamer, into the tPERTand fusing a corresponding recruiting protein to the prime editor (e.g.,via a linker to the napDNAbp or via a linker to the polymerase) that iscapable of specifically binding to the RNA-protein recruitment domain,thereby recruiting the tPERT molecule to the prime editor complex. Asdepicted in the process described in FIG. 3H, the RP-PE:gRNA complexbinds to and nicks the target DNA sequence. Then, the recruiting protein(RP) recruits a tPERT to co-localize to the prime editor complex boundto the DNA target site, thereby allowing the primer binding site,located on the tPERT, to bind to the primer sequence on the nickedstrand, and subsequently, allowing the polymerase (e.g., RT) tosynthesize a single strand of DNA against the DNA synthesis template,located on the tPERT, up through the 5′ end of the tPERT.

While the tPERT is shown in FIG. 3G and FIG. 3H as comprising the PBSand DNA synthesis template on the 5′ end of the RNA-protein recruitmentdomain, the tPERT in other configurations may be designed with the PBSand DNA synthesis template located on the 3′ end of the RNA-proteinrecruitment domain. However, the tPERT with the 5′ extension has theadvantage that synthesis of the single strand of DNA will naturallyterminate at the 5′ end of the tPERT and thus, does not risk using anyportion of the RNA-protein recruitment domain as a template during theDNA synthesis stage of prime editing.

PEgRNA Design Method

The present disclosure also relates to methods for designing PEgRNAs.

In one aspect of design, the design approach can take into account theparticular application for which prime editing is being used. Forinstance, and as exemplified and discussed herein, prime editing can beused, without limitation, to (a) install mutation-correcting changes toa nucleotide sequence, (b) install protein and RNA tags, (c) installimmunoepitopes on proteins of interest, (d) install inducibledimerization domains in proteins, (e) install or remove sequences toalter that activity of a biomolecule, (f) install recombinase targetsites to direct specific genetic changes, and (g) mutagenesis of atarget sequence by using an error-prone RT. In addition to these methodswhich, in general, insert, change, or delete nucleotide sequences attarget sites of interest, prime editors can also be used to constructhighly programmable libraries, as well as to conduct cell data recordingand lineage tracing studies. In these various uses, there may be asdescribed herein particular design aspects pertaining to the preparationof a PEgRNA that is particularly useful for any given of theseapplications.

When designing a PEgRNA for any particular application or use of primeediting, a number of considerations may be taken into account, whichinclude, but are not limited to:

-   -   (a) the target sequence, i.e., the nucleotide sequence in which        one or more nucleobase modifications are desired to be installed        by the prime editor;    -   (b) the location of the cut site within the target sequence,        i.e., the specific nucleobase position at which the prime editor        will induce a single-stand nick to create a 3′ end RT primer        sequence on one side of the nick and the 5′ end endogenous flap        on the other side of the nick (which ultimately is removed by        FEN1 or equivalent thereto and replaced by the 3′ ssDNA flap.        The cut site is analogous to the “edit location” since this what        creates the 3′ end RT primer sequence which becomes extended by        the RT during RNA-depending DNA polymerization to create the 3′        ssDNA flap containing the desired edit, which then replaces the        5′ endogenous DNA flap in the target sequence.    -   (c) the available PAM sequences (including the canonical SpCas9        PAM sites, as well as non-canonical PAM sites recognized by Cas9        variants and equivalents with expanded or differing PAM        specificities);    -   (d) the spacing between the available PAM sequences and the        location of the cut site in the target sequence;    -   (e) the particular Cas9, Cas9 variant, or Cas9 equivalent of the        prime editor being used;    -   (f) the sequence and length of the primer binding site;    -   (g) the sequence and length of the edit template;    -   (h) the sequence and length of the homology arm;    -   (i) the spacer sequence and length; and    -   (j) the core sequence.

The instant disclosure discusses these aspects above.

In one embodiment, an approach to designing a suitable PEgRNA, andoptionally a nicking-sgRNA design guide for second-site nicking, ishereby provided. This embodiment provides a step-by-step set ofinstructions for designing PEgRNAs and nicking-sgRNAs for prime editingwhich takes into account one or more of the above considerations. Thesteps reference the examples shown in FIGS. 70A-70I.

-   -   1. Define the target sequence and the edit. Retrieve the        sequence of the target DNA region (˜200 bp) centered around the        location of the desired edit (point mutation, insertion,        deletion, or combination thereof). See FIG. 70A.    -   2. Locate target PAMs. Identify PAMs in the proximity to the        desired edit location. PAMs can be identified on either strand        of DNA proximal to the desired edit location. While PAMs close        to the edit position are preferred (i.e., wherein the nick site        is less than 30 nt from the edit position, or less than 29 nt,        28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt,        19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt,        10 nt, 9 nt, 8 nt, 7 nt, 6 nt, 5 nt, 4 nt, 3 nt, or 2 nt from        the edit position to the nick site), it is possible to install        edits using protospacers and PAMs that place the nick ≥30 nt        from the edit position. See FIG. 70B.    -   3. Locate the nick sites. For each PAM being considered,        identify the corresponding nick site and on which strand. For Sp        Cas9 H840A nickase, cleavage occurs in the PAM-containing strand        between the 3^(rd) and 4^(th) bases 5′ to the NGG PAM. All        edited nucleotides must exist 3′ of the nick site, so        appropriate PAMs must place the nick 5′ to the target edit on        the PAM-containing strand. In the example shown below, there are        two possible PAMs. For simplicity, the remaining steps will        demonstrate the design of a PEgRNA using PAM 1 only. See FIG.        70C.    -   4. Design the spacer sequence. The protospacer of Sp Cas9        corresponds to the 20 nucleotides 5′ to the NGG PAM on the        PAM-containing strand. Efficient Pol III transcription        initiation requires a G to be the first transcribed nucleotide.        If the first nucleotide of the protospacer is a G, the spacer        sequence for the PEgRNA is simply the protospacer sequence. If        the first nucleotide of the protospacer is not a G, the spacer        sequence of the PEgRNA is G followed by the protospacer        sequence. See FIG. 70D.    -   5. Design a primer binding site (PBS). Using the starting allele        sequence, identify the DNA primer on the PAM-containing strand.        The 3′ end of the DNA primer is the nucleotide just upstream of        the nick site (i.e. the 4^(h) base 5′ to the NGG PAM for Sp        Cas9). As a general design principle for use with PE2 and PE3, a        PEgRNA primer binding site (PBS) containing 12 to 13 nucleotides        of complementarity to the DNA primer can be used for sequences        that contain ˜40-60% GC content. For sequences with low GC        content, longer (14- to 15-nt) PBSs should be tested. For        sequences with higher GC content, shorter (8- to 11-nt) PBSs        should be tested. Optimal PBS sequences should be determined        empirically, regardless of GC content. To design a length-p PBS        sequence, take the reverse complement of the first p nucleotides        5′ of the nick site in the PAM-containing strand using the        starting allele sequence. See FIG. 70E.    -   6. Design an RT template (or DNA synthesis template). The RT        template (or DNA synthesis template where the polymerase is not        reverse transcriptase) encodes the designed edit and homology to        the sequence adjacent to the edit. In one embodiment, these        regions correspond to the DNA synthesis template of FIG. 3D and        FIG. 3E, wherein the DNA synthesis template comprises the “edit        template” and the “homology arm.” Optimal RT template lengths        vary based on the target site. For short-range edits (positions        +1 to +6), it is recommended to test a short (9 to 12 nt), a        medium (13 to 16 nt), and a long (17 to 20 nt) RT template. For        long-range edits (positions +7 and beyond), it is recommended to        use RT templates that extend at least 5 nt (preferably 10 or        more nt) past the position of the edit to allow for sufficient        3′ DNA flap homology. For long-range edits, several RT templates        should be screened to identify functional designs. For larger        insertions and deletions (≥5 nt), incorporation of greater 3′        homology (˜20 nt or more) into the RT template is recommended.        Editing efficiency is typically impaired when the RT template        encodes the synthesis of a G as the last nucleotide in the        reverse transcribed DNA product (corresponding to a C in the RT        template of the PEgRNA). As many RT templates support efficient        prime editing, avoidance of G as the final synthesized        nucleotide is recommended when designing RT templates. To design        a length-r RT template sequence, use the desired allele sequence        and take the reverse complement of the first r nucleotides 3′ of        the nick site in the strand that originally contained the PAM.        Note that compared to SNP edits, insertion or deletion edits        using RT templates of the same length will not contain identical        homology. See FIG. 70F.    -   7. Assemble the full PEgRNA sequence. Concatenate the PEgRNA        components in the following order (5′ to 3′): spacer, scaffold,        RT template and PBS. See FIG. 70G.    -   8. Designing nicking-sgRNAs for PE3. Identify PAMs on the        non-edited strand upstream and downstream of the edit. Optimal        nicking positions are highly locus-dependent and should be        determined empirically. In general, nicks placed 40 to 90        nucleotides 5′ to the position across from the PEgRNA-induced        nick lead to higher editing yields and fewer indels. A nicking        sgRNA has a spacer sequence that matches the 20-nt protospacer        in the starting allele, with the addition of a 5′-G if the        protospacer does not begin with a G. See FIG. 70H.    -   9. Designing PE3b nicking-sgRNAs. If a PAM exists in the        complementary strand and its corresponding protospacer overlaps        with the sequence targeted for editing, this edit could be a        candidate for the PE3b system. In the PE3b system, the spacer        sequence of the nicking-sgRNA matches the sequence of the        desired edited allele, but not the starting allele. The PE3b        system operates efficiently when the edited nucleotide(s) falls        within the seed region (˜10 nt adjacent to the PAM) of the        nicking-sgRNA protospacer. This prevents nicking of the        complementary strand until after installation of the edited        strand, preventing competition between the PEgRNA and the sgRNA        for binding the target DNA. PE3b also avoids the generation of        simultaneous nicks on both strands, thus reducing indel        formation significantly while maintaining high editing        efficiency. PE3b sgRNAs should have a spacer sequence that        matches the 20-nt protospacer in the desired allele, with the        addition of a 5′ G if needed. See FIG. 70I.

The above step-by-step process for designing a suitable PEgRNA and asecond-site nicking sgRNA is not meant to be limiting in any way. Thedisclosure contemplates variations of the above-described step-by-stepprocess which would be derivable therefrom by a person of ordinary skillin the art.

[7] Applications Utilizing Prime Editing

In addition to the development of the prime editing system describedherein as a new “search-and-replace” genome editing technology thatmediates targeted insertions, deletions, and all 12 possiblebase-to-base conversions at targeted loci in human cells withoutrequiring double-stranded DNA breaks, or donor DNA templates, theinventors have also contemplated the use of the prime editors in awide-array of specific applications. For example, and as exemplified anddiscussed herein, prime editing can be used to (a) installmutation-correcting changes to a nucleotide sequence, (b) installprotein and RNA tags, (c) installation of immunoepitopes on proteins ofinterest, (d) install inducible dimerization domains in proteins, (e)install or remove sequences to alter that activity of a biomolecule, (f)install recombinase target sites to direct specific genetic changes, and(g) mutagenesis of a target sequence by using an error-prone RT. Inaddition to these methods which, in general, insert, change, or deletenucleotide sequences at target sites of interest, prime editors can alsobe used to construct highly programmable libraries, as well as toconduct cell data recording and lineage tracing studies. The inventorshave also contemplated additional design features of PEgRNAs that areaimed to improve the efficacy of prime editing. Still further, theinventors have conceived of methods for successfully delivering primeeditors using vector delivery systems and which involve splitting thenapDNAbp using intein domains.

These specific exemplary uses of prime editing are in no way intended tobe limiting. The present Application contemplates any use for primeediting which involves, in general, some form of the installation,removal, and/or modification of one or more nucleobases at a target sitein a nucleotide sequence, e.g., a genomic DNA.

For any of the exemplified uses for prime editing, one may use any primeeditor disclosed herein, including PE1, PE2, PE3, and PE3b, or PE-short.

A. Prime Editing Mechanism

In various embodiments, prime editing (or “prime editing”) operates bycontacting a target DNA molecule (for which a change in the nucleotidesequence is desired to be introduced) with a nucleic acid programmableDNA binding protein (napDNAbp) complexed with an extended guide RNA. Inreference to FIG. 1G, the extended guide RNA comprises an extension atthe 3′ or 5′ end of the guide RNA, or at an intramolecular location inthe guide RNA and encodes the desired nucleotide change (e.g., singlenucleotide change, insertion, or deletion). In step (a), thenapDNAbp/extended gRNA complex contacts the DNA molecule and theextended gRNA guides the napDNAbp to bind to a target locus. In step(b), a nick in one of the strands of DNA of the target locus isintroduced (e.g., by a nuclease or chemical agent), thereby creating anavailable 3′ end in one of the strands of the target locus. In certainembodiments, the nick is created in the strand of DNA that correspondsto the R-loop strand, i.e., the strand that is not hybridized to theguide RNA sequence, i.e., the “non-target strand.” The nick, however,could be introduced in either of the strands. That is, the nick could beintroduced into the R-loop “target strand” (i.e., the strand hybridizedto the protospacer sequence of the extended gRNA) or the “non-targetstrand” (i.e, the strand forming the single-stranded portion of theR-loop and which is complementary to the target strand). In step (c),the 3′ end of the DNA strand (formed by the nick) interacts with theextended portion of the guide RNA in order to prime reversetranscription (i.e, “target-primed RT”). In certain embodiments, the 3′end DNA strand hybridizes to a specific RT priming sequence on theextended portion of the guide RNA, i.e, the “reverse transcriptasepriming sequence.” In step (d), a reverse transcriptase is introduced(as a fusion protein with the napDNAbp or in trans) which synthesizes asingle strand of DNA from the 3′ end of the primed site towards the 5′end of the extended guide RNA. This forms a single-strand DNA flapcomprising the desired nucleotide change (e.g., the single base change,insertion, or deletion, or a combination thereof) and which is otherwisehomologous to the endogenous DNA at or adjacent to the nick site. Instep (e), the napDNAbp and guide RNA are released. Steps (f) and (g)relate to the resolution of the single strand DNA flap such that thedesired nucleotide change becomes incorporated into the target locus.This process can be driven towards the desired product formation byremoving the corresponding 5′ endogenous DNA flap (e.g., by FEN1 orsimilar enzyme that is provide in trans, as a fusion with the primeeditor, or endogenously provided) that forms once the 3′ single strandDNA flap invades and hybridizes to the endogenous DNA sequence. Withoutbeing bound by theory, the cells endogenous DNA repair and replicationprocesses resolves the mismatched DNA to incorporate the nucleotidechange(s) to form the desired altered product. The process can also bedriven towards product formation with “second strand nicking,” asexemplified in FIG. 1G, or “temporal second strand nicking,” asexemplified in FIG. 1I and discussed herein.

The process of prime editing may introduce at least one or more of thefollowing genetic changes: transversions, transitions, deletions, andinsertions. In addition, prime editing may be implemented for specificapplications. For example, and as exemplified and discussed herein,prime editing can be used to (a) install mutation-correcting changes toa nucleotide sequence, (b) install protein and RNA tags, (c)installation of immunoepitopes on proteins of interest, (d) installinducible dimerization domains in proteins, (e) install or removesequences to alter that activity of a biomolecule, (f) installrecombinase target sites to direct specific genetic changes, and (g)mutagenesis of a target sequence by using an error-prone RT. In additionto these methods which, in general, insert, change, or delete nucleotidesequences at target sites of interest, prime editors can also be used toconstruct highly programmable libraries, as well as to conduct cell datarecording and lineage tracing studies. The inventors have alsocontemplated additional design features of PEgRNAs that are aimed toimprove the efficacy of prime editing. Still further, the inventors haveconceived of methods for successfully delivering prime editors usingvector delivery systems and which involve splitting the napDNAbp usingintein domains.

The term “prime editing system” or “prime editor (PE)” refers thecompositions involved in the method of genome editing usingtarget-primed reverse transcription (TPRT) describe herein, including,but not limited to the napDNAbps, reverse transcriptases, fusionproteins (e.g., comprising napDNAbps and reverse transcriptases),extended guide RNAs, and complexes comprising fusion proteins andextended guide RNAs, as well as accessory elements, such as secondstrand nicking components and 5′ endogenous DNA flap removalendonucleases (e.g., FEN1) for helping to drive the prime editingprocess towards the edited product formation.

In another embodiment, the schematic of FIG. 3F depicts the interactionof a typical PEgRNA with a target site of a double stranded DNA and theconcomitant production of a 3′ single stranded DNA flap containing thegenetic change of interest. The double strand DNA is shown with the topstrand in the 3′ to 5′ orientation and the lower strand in the 5′ to 3′direction. The top strand comprises the “protospacer” and the PAMsequence and is referred to as the “target strand.” The complementarylower strand is referred to as the “non-target strand.” Although notshown, the PEgRNA depicted would be complexed with a Cas9 or equivalent.As shown in the schematic, the spacer of the PEgRNA anneals to acomplementary region on the target strand, which is referred to as theprotospacer, which is located just downstream of the PAM sequence isapproximately 20 nucleotides in length. This interaction forms asDNA/RNA hybrid between the spacer RNA and the protospacer DNA, andinduces the formation of an R loop in the region opposite theprotospacer. As taught elsewhere herein, the Cas9 protein (not shown)then induces a nick in the non-target strand, as shown. This then leadsto the formation of the 3′ ssDNA flap region which, in accordance with*z*, interacts with the 3′ end of the PEgRNA at the primer binding site.The 3′ end of the ssDNA flap (i.e., the reverse transcriptase primersequence) anneals to the primer binding site (A) on the PEgRNA, therebypriming reverse transcriptase. Next, reverse transcriptase (e.g.,provided in trans or provided cis as a fusion protein, attached to theCas9 construct) then polymerizes a single strand of DNA which is codedfor by the edit template (B) and homology arm (C). The polymerizationcontinues towards the 5′ end of the extension arm. The polymerizedstrand of ssDNA forms a ssDNA 3′ end flap which, as describe elsewhere(e.g., as shown in FIG. 1G), invades the endogenous DNA, displacing thecorresponding endogenous strand (which is removed as a 5′ DNA flap ofendogenous DNA), and installing the desired nucleotide edit (singlenucleotide base pair change, deletions, insertions (including wholegenes) through naturally occurring DNA repair/replication rounds.

This application of prime editing can be further described in Example 1.

B. Mutagenesis Using Prime Editing with Error-Drone RT

In various embodiments, the prime editing system (i.e., prime editingsystem) may include the use of an error-prone reverse transcriptase forperforming targeted mutagenesis, i.e., to mutate only a well-definedstretch of DNA in a genome or other DNA element in a cell. FIG. 22provides a schematic of an exemplary process for introducing conductingtargeted mutagenesis with an error-prone reverse transcriptase on atarget locus using a nucleic acid programmable DNA binding protein(napDNAbp) complexed with an extended guide RNA. This process may bereferred to as an embodiment of prime editing for targeted mutagenesis.The extended guide RNA comprises an extension at the 3′ or 5′ end of theguide RNA, or at an intramolecular location in the guide RNA. In step(a), the napDNAbp/gRNA complex contacts the DNA molecule and the gRNAguides the napDNAbp to bind to the target locus to be mutagenized. Instep (b), a nick in one of the strands of DNA of the target locus isintroduced (e.g., by a nuclease or chemical agent), thereby creating anavailable 3′ end in one of the strands of the target locus. In certainembodiments, the nick is created in the strand of DNA that correspondsto the R-loop strand, i.e., the strand that is not hybridized to theguide RNA sequence. In step (c), the 3′ end DNA strand interacts withthe extended portion of the guide RNA in order to prime reversetranscription. In certain embodiments, the 3′ ended DNA strandhybridizes to a specific RT priming sequence on the extended portion ofthe guide RNA. In step (d), an error-prone reverse transcriptase isintroduced which synthesizes a mutagenized single strand of DNA from the3′ end of the primed site towards the 3′ end of the guide RNA. Exemplarymutations are indicated with an asterisk “*”. This forms a single-strandDNA flap comprising the desired mutagenized region. In step (e), thenapDNAbp and guide RNA are released. Steps (f) and (g) relate to theresolution of the single strand DNA flap (comprising the mutagenizedregion) such that the desired mutagenized region becomes incorporatedinto the target locus. This process can be driven towards the desiredproduct formation by removing the corresponding 5′ endogenous DNA flapthat forms once the 3′ single strand DNA flap invades and hybridizes tothe complementary sequence on the other strand. The process can also bedriven towards product formation with second strand nicking, asexemplified in FIG. 1F. Following endogenous DNA repair and/orreplication processes, the mutagenized region becomes incorporated intoboth strands of DNA of the DNA locus.

This application of prime editing can be further described in Example 2.

Error-prone or mutagenic RT enzymes are known in the art. As usedherein, the term “error-prone” reverse transcriptase refers to a reversetranscriptase enzyme that occurs naturally or which has been derivedfrom another reverse transcriptase (e.g., a wild type M-MLV reversetranscriptase) which has an error rate that is less than the error rateof wild type M-MLV reverse transcriptase. The error rate of wild typeM-MLV reverse transcriptase is reported to be in the range of one errorin 15,000 to 27,000 nucleobase incorporations. See Boutabout et al.(2001) “DNA synthesis fidelity by the reverse transcriptase of the yeastretrotransposon Ty1,” Nucleic Acids Res 29(11):2217-2222, which isincorporated herein by reference. Thus, for purposes of thisapplication, the term “error prone” refers to those RT that have anerror rate that is greater than one error in 15,000 nucleobaseincorporation (6.7 ×10⁻⁵ or higher), e.g., 1 error in 14,000 nucleobases(7.14×10⁻⁵ or higher), 1 error in 13,000 nucleobases or fewer (7.7×10⁻⁵or higher), 1 error in 12,000 nucleobases or fewer (7.7×10⁻⁵ or higher),1 error in 11,000 nucleobases or fewer (9.1×10⁻⁵ or higher), 1 error in10,000 nucleobases or fewer (1×10⁻⁴ or 0.0001 or higher), 1 error in9,000 nucleobases or fewer (0.00011 or higher), 1 error in 8,000nucleobases or fewer (0.00013 or higher) 1 error in 7,000 nucleobases orfewer (0.00014 or higher), 1 error in 6,000 nucleobases or fewer(0.00016 or higher), 1 error in 5,000 nucleobases or fewer (0.0002 orhigher), 1 error in 4,000 nucleobases or fewer (0.00025 or higher), 1error in 3,000 nucleobases or fewer (0.00033 or higher), 1 error in2,000 nucleobase or fewer (0.00050 or higher), or 1 error in 1,000nucleobases or fewer (0.001 or higher), or 1 error in 500 nucleobases orfewer (0.002 or higher), or 1 error in 250 nucleobases or fewer (0.004or higher).

A variety of mutagenic RTs could be envisioned for generation ofmutagenized sequences using prime editing. Two such examples are themutagenic reverse transcriptases from Bordetella phage (see Handa, S.,et al. Nucl Acids Res 9711-25 (2018), which is incorporated herein byreference) and Legionella pneumophila (see Arambula, D., et al. ProcNatl Acad Sci USA 8212-7 (2013), which is incorporated by reference). Inthe case of the RT from Bordetella phage (brt), an accessory proteinmight need to also be added (bavd) to Cas9—or delivered in trans—as wellas additional RNA sequences to the PEgRNA to improve binding of themutagenic RT to the target site (see Handa, S., et al. Nucl Acids Res9711-25 (2018)). When using mutagenic RTs, the template region of thePEgRNA might be enriched in adenosines or AAY codons to enhancediversity.

The amino acid sequence of the mutagenic RT from Bordetella phage isprovided as follows. Like other RTs disclosed herein, the Brt proteinmay be fused to a napDNAbp as a fusion protein to form a functional PE.

Name Sequence brt MGKRHRNLIDQITTWENLLDAYRKTSHGKRRTWGYLEFKEY mutagenicDLANLLALQAELKAGNYERGPYREFLVYEPKPRLISALEFK rtDRLVQHALCNIVAPIFEAGLLPYTYACRPDKGTHAGVCHVQAELRRTRATHFLKSDFSKFFPSIDRAALYAMIDKKIHCAATRRLLRVVLPDEGVGIPIGSLTSQLFANVYGGAVDRLLHDELKQRHWARYMDDIVVLGDDPEELRAVFYRLRDFASERLGLKISHWQVAPVSRGINFLGYRIWPTHKLLRKSSVKRAKRKVANFIKHGEDESLQRFLASWSGHAQWADTHNLFTWMEEQYGIACH (SEQ ID NO: 235)

In the case of Brt from Bordetella, the PE fusion may also include anadditional accessory protein (Bavd). The accessory protein may be fusedto the PE fusion protein or provided in trans. The amino acid sequenceof Bavd accessory protein is provided as follows:

Name Sequence bavd MEPIEEATKCYDQMLIVERYERVISYLYPIAQSIP accessory RKHGVAREMFLKCLLGQVELFIVAGKSNQVSKLYA proteinADAGLAMLRFWLRFLAGIQKPHAMTPHQVETAQVL to brtIAEVGRILGSWIARVNRKGQAGK (SEQ ID NO: 236)

In the case of Brt from Bordetella, the PEgRNA may comprise anadditional nucleotide sequence added a PEgRNA, e.g., to the 5′ or 3′end. Exemplary sequence is as follows, which is originally from theBordetella phage genome:

NAME SEQUENCE PEGRNA- ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGG ADDITIONCUGGCCUUUCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUG 1CUUCGGCGAACACGUUACACGGUUCGGCAAAACGUCGAUUACUGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCU GGGAAAUAA (SEQ ID NO: 237)

This PEgRNA addition sequence can be reduced in various ways to shortenthe length. For example, the PEgRNA-addition 1 sequence could be reducedto the following exemplary alternative addition sequences:

NAME SEQUENCE PEGRNA- ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCADDITION CUUUCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGCGAA 2CACGUUACACGGUUCGGCAAAACGUCGAUUACUGAAAAUGGAAAGGCGGGGCCGACUUC (SEQ ID NO: 238) PEGRNA-ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGC ADDITIONCUUUCCUCAAUAGGUGGUCAAAGGGCAGGCUGGGAAAUAA (SEQ 3 ID NO: 239) PEGRNA-ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGC ADDITIONCUUUCCUCAAUAGGUGGUCA (SEQ ID NO: 277) 4 PEGRNA-CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUUCCUCA ADDITIONAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGCGAACACGUUACA 5CGGUUCGGCAAAACGUCGAUUACUGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCUGGGAAAUAA (SEQ ID NO: 240) PEGRNA-CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUUCCUCA ADDITIONAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGCGAACACGUUACA 6CGGUUCGGCAAAACGUCGAUUACUGAAAAUGGAAAGGCGGGGCCG ACUUC (SEQ ID NO: 241)PEGRNA- CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUUCCUCA ADDITIONAUAGGUGGUCAAAGGGCAGGCUGGGAAAUAA (SEQ ID NO: 242) 7 PEGRNA-CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUUCCUCA ADDITION AUAGGUGGUCA 8(SEQ ID NO: 243)

In other embodiments, the PEgRNA addition sequence can be also bemutated. For example, the PEgRNA-addition 1 sequence could be mutated tothe following exemplary alternative addition sequence:

NAME SEQUENCE PEGRNA- ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGC ADDITIONUGGCCUUUCCUCAAUAGAUGAGCCGCCGGUUCUGUCCUGCU 1UCGGCGAACACGUUACACGGUUCGGCAAAACGUCGAUUACU MUTATEDGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCUGGGA AAUAA (SEQ ID NO: 244)

In various embodiments relating to the use of PE for introducingmutations, special PEgRNA considerations may apply. For example, withoutwishing to be bound by theory, the additional PEgRNA sequences describedabove might be needed to enable efficient mutagenesis via mutagenic RTs.

Any mutagenic RT may be used with the prime editors disclosed herein.For example, the error-prone RT described in the following referencesmay be used and are incorporated herein by reference:

-   Bebenek et al., “Error-prone polymerization by HIV-1 reverse    transcriptase. Contribution of template-primer misalignment,    miscoding, and termination probability to mutational hot spots.,” J.    Biol Chem, 1993, 268: 10324-34; and-   Menendez-Arias, “Mutation rates and instrinsic fidelity of    retroviral reverse transcriptases,” 2009, Viruses, 1(3): 1137-1165.

Various error-prone RTs can include, but are not limited to, thefollowing enzymes disclosed in Table 1 of Menendez-Arias et al. (theentire contents of the reference of which are incorporated byreference), as follows:

ERROR-PRONE RT REPORTED ERROR-RATE RANGE HIV-1 RT (GROUP M, 0.6 × 10-4TO 2.0 × 10-4 SUBTYPE B) HIV-1 RT (GROUP O) 5.5 × 10-5 SIV AGM RT 2.9 ×10-5 SIV MNE RT 1.6 × 10-5 TO 1.2 × 10-4 PFV RT 1.7 × 10-4 FIV RT 6.2 ×10-5 AMV RT 5.9 × 10-5 MO-MLV RT 2.7 × 10-5 TO 3.3 × 10-5

C. Use of Prime Editing for Treating Triplet Expansion Disorders

The prime editing system or prime editing (PE) system described hereinmay be used to contract trinucleotide repeat mutations (or “tripletexpansion diseases”) to treating conditions such as Huntington's diseaseand other trinucleotide repeat disorders. Trinucleotide repeat expansiondisorders are complex, progressive disorders that involve developmentalneurobiology and often affect cognition as well as sensori-motorfunctions. The disorders show genetic anticipation (i.e. increasedseverity with each generation). The DNA expansions or contractionsusually happen meiotically (i.e. during the time of gametogenesis, orearly in embryonic development), and often have sex-bias meaning thatsome genes expand only when inherited through the female, others onlythrough the male. In humans, trinucleotide repeat expansion disorderscan cause gene silencing at either the transcriptional or translationallevel, which essentially knocks out gene function. Alternatively,trinucleotide repeat expansion disorders can cause altered proteinsgenerated with large repetitive amino acid sequences that eitherabrogate or change protein function, often in a dominant-negative manner(e.g. poly-glutamine diseases).

Without wishing to be bound by theory, triplet expansion is caused byslippage during DNA replication or during DNA repair synthesis. Becausethe tandem repeats have identical sequence to one another, base pairingbetween two DNA strands can take place at multiple points along thesequence. This may lead to the formation of “loop out” structures duringDNA replication or DNA repair synthesis. This may lead to repeatedcopying of the repeated sequence, expanding the number of repeats.Additional mechanisms involving hybrid RNA:DNA intermediates have beenproposed. Prime editing may be used to reduce or eliminate these tripletexpansion regions by deletion one or more or the offending repeat codontriplets. In an embodiment of this use, FIG. 23 , provides a schematicof a PEgRNA design for contracting or reducing trinucleotide repeatsequences with prime editing.

Prime editing may be implemented to contract triplet expansion regionsby nicking a region upstream of the triplet repeat region with the primeeditor comprising a PEgRNA appropriated targeted to the cut site. Theprime editor then synthesizes a new DNA strand (ssDNA flap) based on thePEgRNA as a template (i.e., the edit template thereof) that codes for ahealthy number of triplet repeats (which depends on the particular geneand disease). The newly synthesized ssDNA strand comprising the healthytriplet repeat sequence also is synthesized to include a short stretchof homology (i.e., the homology arm) that matches the sequence adjacentto the other end of the repeat. Invasion of the newly synthesizedstrand, and subsequent replacement of the endogenous DNA with the newlysynthesized ssDNA flap, leads to a contracted repeat allele.

Depending on the particular trinucleotide expansion disorder, thedefect-inducing triplet expansions may occur in “trinucleotide repeatexpansion proteins.” Trinucleotide repeat expansion proteins are adiverse set of proteins associated with susceptibility for developing atrinucleotide repeat expansion disorder, the presence of a trinucleotiderepeat expansion disorder, the severity of a trinucleotide repeatexpansion disorder or any combination thereof. Trinucleotide repeatexpansion disorders are divided into two categories determined by thetype of repeat. The most common repeat is the triplet CAG, which, whenpresent in the coding region of a gene, codes for the amino acidglutamine (Q). Therefore, these disorders are referred to as thepolyglutamine (polyQ) disorders and comprise the following diseases:Huntington Disease (HD); Spinobulbar Muscular Atrophy (SBMA);Spinocerebellar Ataxias (SCA types 1, 2, 3, 6, 7, and 17); andDentatorubro-Pallidoluysian Atrophy (DRPLA). The remaining trinucleotiderepeat expansion disorders either do not involve the CAG triplet or theCAG triplet is not in the coding region of the gene and are, therefore,referred to as the non-polyglutamine disorders. The non-polyglutaminedisorders comprise Fragile X Syndrome (FRAXA); Fragile XE MentalRetardation (FRAXE); Friedreich Ataxia (FRDA); Myotonic Dystrophy (DM);and Spinocerebellar Ataxias (SCA types 8, and 12).

The proteins associated with trinucleotide repeat expansion disorderscan be selected based on an experimental association of the proteinassociated with a trinucleotide repeat expansion disorder to atrinucleotide repeat expansion disorder. For example, the productionrate or circulating concentration of a protein associated with atrinucleotide repeat expansion disorder may be elevated or depressed ina population having a trinucleotide repeat expansion disorder relativeto a population lacking the trinucleotide repeat expansion disorder.Differences in protein levels may be assessed using proteomic techniquesincluding but not limited to Western blot, immunohistochemical staining,enzyme linked immunosorbent assay (ELISA), and mass spectrometry.Alternatively, the proteins associated with trinucleotide repeatexpansion disorders may be identified by obtaining gene expressionprofiles of the genes encoding the proteins using genomic techniquesincluding but not limited to DNA microarray analysis, serial analysis ofgene expression (SAGE), and quantitative real-time polymerase chainreaction (Q-PCR).

Non-limiting examples of proteins associated with trinucleotide repeatexpansion disorders which can be corrected by prime editing include AR(androgen receptor), FMR1 (fragile X mental retardation 1), HTT(huntingtin), DMPK (dystrophia myotonica-protein kinase), FXN(frataxin), ATXN2 (ataxin 2), ATN1 (atrophin 1), FEN1 (flapstructure-specific endonuclease 1), TNRC6A (trinucleotide repeatcontaining 6A), PABPN1 (poly(A) binding protein, nuclear 1), JPH3(junctophilin 3), MED15 (mediator complex subunit 15), ATXN1 (ataxin 1),ATXN3 (ataxin 3), TBP (TATA box binding protein), CACNA1A (calciumchannel, voltage-dependent, P/Q type, alpha 1A subunit), ATXN80S (ATXN8opposite strand (non-protein coding)), PPP2R2B (protein phosphatase 2,regulatory subunit B, beta), ATXN7 (ataxin 7), TNRC6B (trinucleotiderepeat containing 6B), TNRC6C (trinucleotide repeat containing 6C),CELF3 (CUGBP, Elav-like family member 3), MAB21L1 (mab-21-like 1 (C.elegans)), MSH2 (mutS homolog 2, colon cancer, nonpolyposis type 1 (E.coli)), TMEM185A (transmembrane protein 185A), SIX5 (SIX homeobox 5),CNPY3 (canopy 3 homolog (zebrafish)), FRAXE (fragile site, folic acidtype, rare, fra(X)(q28) E), GNB2 (guanine nucleotide binding protein (Gprotein), beta polypeptide 2), RPL14 (ribosomal protein L14), ATXN8(ataxin 8), INSR (insulin receptor), TTR (transthyretin), EP400 (E1Abinding protein p400), GIGYF2 (GRB10 interacting GYF protein 2), OGG1(8-oxoguanine DNA glycosylase), STC1 (stanniocalcin 1), CNDP1 (carnosinedipeptidase 1 (metallopeptidase M20 family)), C10orf2 (chromosome 10open reading frame 2), MAML3 mastermind-like 3 (Drosophila), DKC1(dyskeratosis congenita 1, dyskerin), PAXIP1 (PAX interacting (withtranscription-activation domain) protein 1), CASK(calcium/calmodulin-dependent serine protein kinase (MAGUK family)),MAPT (microtubule-associated protein tau), SP1 (Sp1 transcriptionfactor), POLG (polymerase (DNA directed), gamma), AFF2 (AF4/FMR2 family,member 2), THBS1 (thrombospondin 1), TP53 (tumor protein p53), ESR1(estrogen receptor 1), CGGBP1 (CGG triplet repeat binding protein 1),ABT1 (activator of basal transcription 1), KLK3 (kallikrein-relatedpeptidase 3), PRNP (prion protein), JUN (jun oncogene), KCNN3 (potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 3), BAX (BCL2-associated X protein), FRAXA (fragile site, folicacid type, rare, fra(Xxq27.3) A (macroorchidism, mental retardation)),KBTBD10 (kelch repeat and BTB (POZ) domain containing 10), MBNL1(muscleblind-like (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, E.coli) (S. cerevisiae)), NCOA3 (nuclear receptor coactivator 3), ERDA1(expanded repeat domain, CAG/CTG 1), TSC1 (tuberous sclerosis 1), COMP(cartilage oligomeric matrix protein), GCLC (glutamate-cysteine ligase,catalytic subunit), RRAD (Ras-related associated with diabetes), MSH3(mutS homolog 3 (E. coli)), DRD2 (dopamine receptor D2), CD44 (CD44molecule (Indian blood group)), CTCF (CCCTC-binding factor (zinc fingerprotein)), CCND1 (cyclin D1), CLSPN (claspin homolog (Xenopus laevis)),MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase,receptor type, U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase),TRIM22 (tripartite motif-containing 22), WT1 (Wilms tumor 1), AHR (arylhydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurineS-methyltransferase), NDP (Norrie disease (pseudoglioma)), ARX(aristaless related homeobox), MUS81 (MUS81 endonuclease homolog (S.cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (earlygrowth response 1), UNG (uracil-DNA glycosylase), NUMBL (numb homolog(Drosophila)-like), FABP2 (fatty acid binding protein 2, intestinal),EN2 (engrailed homeobox 2), CRYGC (crystallin, gamma C), SRP14 (signalrecognition particle 14 kDa (homologous Alu RNA binding protein)), CRYGB(crystallin, gamma B), PDCD1 (programmed cell death 1), HOXA1 (homeoboxA1), ATXN2L (ataxin 2-like), PMS2 (PMS2 postmeiotic segregationincreased 2 (S. cerevisiae)), GLA (galactosidase, alpha), CBL (Cas-Br-M(murine) ecotropic retroviral transforming sequence), FTH1 (ferritin,heavy polypeptide 1), IL12RB2 (interleukin 12 receptor, beta 2), OTX2(orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNAdirected), gamma 2, accessory subunit), DLX2 (distal-less homeobox 2),SIRPA (signal-regulatory protein alpha), OTX1 (orthodenticle homeobox1), AHRR (aryl-hydrocarbon receptor repressor), MANF (mesencephalicastrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein158 (gene/pseudogene)), and ENSG00000078687.

The prime editors herein disclosed may be used to contract tripletrepeat expansion regions in any of the above-indicated disease proteins,including following polyglutamine triplet expansion disease genes (whichshow the particular location of the pathogenic repeats that may beremoved wholly or in part by prime editing):

POSITION OF TRIPLET EXPANSION AFFECTED PATHOGENIC DISEASE GENE REPEATSDENTATORUBRO- ATN1 49-88 PALLIDOLUYSIAN ATROPHIN-1 ATROPHY HUNTINGTON'SDISEASE HTT  36-250 THE HUNTINGTIN GENE SPINAL AND BULBAR AR 38-62MUSCULAR ATROPHY ANDROGEN RECEPTOR SPINOCEREBELLAR ATXN1 49-88 ATAXIATYPE 1 ATAXIN 1 SPINOCEREBELLAR ATXN2 33-77 ATAXIA TYPE 2 ATAXIN 2SPINOCEREBELLAR ATXN3 55-86 ATAXIA TYPE 3 ATAXIN 3 SPINOCEREBELLARCACNA1A 21-30 ATAXIA TYPE 6 SPINOCEREBELLAR ATXN7  38-120 ATAXIA TYPE 7ATAXIN 7 SPINOCEREBELLAR TBP 47-63 ATAXIA TYPE 17 TATA-BINDING PROTEIN

The prime editors herein disclosed may also be used to contract tripletrepeat expansion regions typically found in the followingnon-polyglutamine triplet expansion disease genes:

POSITION OF TRIPLET EXPANSION PATHOGENIC DISEASE AFFECTED GENE REPEATSFRAXA (FRAGILE X FMR1 230+ SYNDROME) FRAGILE X MENTAL RETARDATIONPROTEIN FXTAS (FRAGILE X- FMR1  55-200 ASSOCIATED TREMOR/ATAXIASYNDROME) FRAXE (FRAGILE XE AFF2 200+ MENTAL RETARDATION) FRDA(FRIEDREICH'S FXN 100+ ATAXIA) FRATAXIN DM1 (MYOTONIC DMPK  50+DYSTROPHY TYPE 1) MYOTONIN- PROTEIN KINASE SCA8 SCA8 110-250(SPINOCEREBELLAR ATAXIN 8 ATAXIA TYPE 8) SCA12 PPP2R2B 66-78(SPINOCEREBELLAR SERINE/THREONIN ATAXIA TYPE 12) PROTEIN PHOSPHATASE 2A

Prime editing may be implemented to contract triplet expansion regionsusing a PEgRNA with an edit template that is designed to delete at leastone codon of a triplet expansion region. In other embodiments, thePEgRNAs for using in prime editing for this used to delete at least 1,or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or33, or 34, or 35, or 36, or 37, or 38, or 39, or 40, or 41, or 42, or43, or 4, or 45, or 46, or 47, or 48, or 49, or 50, or 51, or 52, or 53,or 54, or 55, or 56, or 57, or 58, or 59, or 60, or 61, or 62, or 63, or64, or 65, or 66, or 67, or 68, or 69, or 70, or 71, or 72, or 73, or74, or 75, or 76, or 77, or 78, or 79, or 80, or 81, or 82, or 83, or84, or 85, or 86, or 87, or 88, or 89, or 90, or 91, or 92, or 93, or94, or 95, or 96, or 97, or 98, or 99, or 100, or more codons from atriplet expansion region in order to arrive at a healthy (i.e., notassociated with producing the disease) number of triplet repeats.

In other embodiments, the PEgRNAs for using in prime editing for thisused to delete at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19,or 20 or more codons from a triplet expansion region in order to arriveat a healthy (i.e., not associated with producing the disease) number oftriplet repeats.

In other embodiments, the PEgRNAs for using in prime editing for thisused to delete at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or9, or 10, or 11, or 12, or 13, or 14, or 15, or more codons from atriplet expansion region in order to arrive at a healthy (i.e., notassociated with producing the disease) number of triplet repeats.

In other embodiments, the PEgRNAs for using in prime editing for thisused to delete at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or9, or 10, or more codons from a triplet expansion region in order toarrive at a healthy (i.e., not associated with producing the disease)number of triplet repeats.

Prime editing may be configured to correct any triplet expansion region,such as those described in Budworth et al., “A Brief History of TripletRepeat Diseases,” Methods Mol Biol, 2013, 1010: 3-17, US 20011/00165540A1 (Genome editing of genes associated with trinucleotide repeatexpansion disorders in animals), US 2016/0355796 A1 (Compositions andmethods of use of crispr-cas systems in nucleotide repeat disorders

In various embodiments, the disclosure provides a prime editingconstruct suitable for use in a cell having a trinucleotide repeatexpansion region in a defective gene comprising (a) a prime editorfusion comprising a napDNAbp and a reverse transcriptase, (b) a PEgRNAcomprising a spacer sequence that targets the trinucleotide repeatexpansion region and an extension arm comprising an edit template thatcodes for the removal of the trinucleotide repeat expansion region.

In various other embodiments, the disclosure provides a method fordeleting all or a portion of a trinucleotide repeat expansion region ina defective gene in a cell using prime editing comprising contacting thecell with a prime editor fusion comprising a napDNAbp and a reversetranscriptase and a PEgRNA comprising a spacer sequence that targets thetrinucleotide repeat expansion region and an extension arm comprising anedit template that codes for the removal of the trinucleotide repeatexpansion region.

In various embodiments, the trinucleotide repeat comprises repeatingCTG, CAG, CGG, CCG, GAA, or TTC trinucleotides.

In various other embodiments, the tetranucleotide repeats,pentanucleotide repeats, or hexanucleotide repeats.

D. Use of Prime Editing for Peptide Tagging

In another aspect, the disclosure provides a method of using the hereindescribed prime editors for genetically grafting one or more peptidetags onto a protein using prime editing, More in particular, thedisclosure provides a method for genetically installing one or morepeptide tags onto a protein comprising: contacting a target nucleotidesequence encoding the protein with a prime editor configured to inserttherein a second nucleotide sequence encoding the one or more peptidetags to result in a recombinant nucleotide sequence that encodes afusion protein comprising the protein fused to the protein tag.

In other embodiments, the disclosure provides a method for making afusion protein comprising a peptide of interest and one or more peptidetags, the method comprising: contacting a target nucleotide sequenceencoding the protein with a prime editor configured to insert therein asecond nucleotide sequence encoding the one or more peptide tags toresult in a recombinant nucleotide sequence that encodes the fusionprotein comprising the protein fused to the protein tag.

In various embodiments, the target nucleotide sequence is a specificgene of interest in a genomic DNA. The gene of interest may encode aprotein of interest (e.g., a receptor, an enzyme, a therapeutic protein,a membrane protein, a transport protein, a signal transduction protein,or an immunological protein, etc.). The gene of interest may also encodean RNA molecule, including, but not limited to, messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA),antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA(siRNA), and cell-free RNA (cfRNA).

The peptide tag may be any peptide tag or variant thereof which impartsone or more functions onto a protein for purposes such as separation,purification, visualization, solubilization, or detection. The peptidestags can include “affinity tags” (to facilitate protein purification),“solubilization tags” (to assist in proper folding of proteins),“chromatography tags” (to alter chromatographic properties of proteins),“epitope tags” (to bind to high affinity antibodies), and “fluorescencetags” (to facilitate visualization of proteins in a cell or in vitro).Examples of peptide tags include, but are not limited to the followingtags:

NAME AMINO ACID SEQUENCE SEQ ID NO: AVITAG ™ GLNDIFEAQKIEWHESEQ ID NO: 245 C-TAG EPEA SEQ ID NO: 246 CALMODULIN-TAGKRRWKKNFIAVSAANRFKKISSSGA SEQ ID NO: 247 L POLYGLUTAMATE TAG EEEEEESEQ ID NO: 248 E-TAG GAPVPYPDPLEPR SEQ ID NO: 249 FLAG-TAG DYKDDDDKSEQ ID NO: 250 HA-TAG YPYDVPDYA SEQ ID NO: 251 HIS-TAG H (HIS₁)SEQ ID NO: 252 HH (HIS₂) SEQ ID NO: 253 HHH (HIS₃) SEQ ID NO: 254HHHH (HIS₄) SEQ ID NO: 255 HHHHH (HIS₅) SEQ ID NO: 256 HHHHHH (HIS₆)SEQ ID NO: 257 HHHHHHH (HIS₇) SEQ ID NO: 258 HHHHHHHH (HIS₈)SEQ ID NO: 259 HHHHHHHHH (HIS₉) SEQ ID NO: 260 HHHHHHHHHH (HIS₁₀)SEQ ID NO: 261 HHHHHHHHHH . . . H . . . (HIS_(N), SEQ ID NO: 262WHEREIN N = 1-25) MYC-TAG EQKLISEEDL SEQ ID NO: 263 NE-TAGTKENPRSNQEESYDDNES SEQ ID NO: 264 RHO1D4-TAG TETSQVAPA SEQ ID NO: 265S-TAG KETAAAKFERQHMDS SEQ ID NO: 266 SBP-TAG MDEKTTGWRGGHVVEGLAGELEQSEQ ID NO: 267 LRARLEHHPQGQREP SOFTAG-1 SLAELLNAGLGGS SEQ ID NO: 268SOFTAG-2 TQDPSRVG SEQ ID NO: 269 SPOT-TAG PDRVRAVSHWSS SEQ ID NO: 270STREP-TAG WSHPQFEK SEQ ID NO: 271 TC TAG CCPGCC SEQ ID NO: 272 TY TAGEVHTNQDPLD SEQ ID NO: 273 V5 TAG GKPIPNPLLGLDST SEQ ID NO: 274 VSV-TAGYTDIEMNRLGK SEQ ID NO: 275 XPRESS TAG DLYDDDDK SEQ ID NO: 276

Peptide tags may also be the following affinity tags (for separationand/or purification of proteins) (as described in Table 9.9.1 of Kimpleet al., “Overview of Affinity Tags for Protein Purification,” CurrProtoc Protein Sci, 2013, 73:Unit-9.9, which is incorporated herein byreference).

NAME AMINO ACID SEQUENCE AU1 EPITOPE DTYRYI SEQ ID NO: 278 AU5 EPITOPETDFYLK SEQ ID NO: 279 BACTERIOPHAGE T7 MASMTGGQQMG SEQ ID NO: 280EPITOPE (T7-TAG) BLUETONGUE VIRUS TAG QYPALT SEQ ID NO: 281 (B-TAG)E2 EPITOPE SSTSSDFRDR SEQ ID NO: 282 HISTIDINE AFFINITY TAGKDHLIHNVHKEFHAHAHNK SEQ ID NO: 283 (HAT) HSV EPITOPE QPELAPEDSEQ ID NO: 284 POLYARGININE RRRRR SEQ ID NO: 285 (ARG-TAG) POLYASPARTATECCCC SEQ ID NO: 286 (ASP-TAG) POLYPHENYLALANINE FFFFFFFFFFFSEQ ID NO: 287 (PHE-TAG) S1-TAG NANNPDWDF SEQ ID NO: 288 S-TAGKETAAAKFERQHMDS SEQ ID NO: 289 VSV-G YTDIEMNRLGK SEQ ID NO: 290

In particular embodiments, the peptide tags may include a His⁶ tag,FLAG-tag, V5-tag, GCN4-tag, HA-tag, Myc-Tag, FIAsH/ReAsH-tag, Sortasesubstrate, pi-clamp.

In various embodiments, the peptide tags may be used for applicationsthat include protein fluorescent labeling, immunoprecipitation,immunoblotting, immunohistochemistry, protein recruitment, inducibleprotein degrons, and genome-wide screening.

In various other embodiments, the peptide tag may include an inteinsequence to install protein self-splicing function. As used herein, theterm “intein” refers to auto-processing polypeptide domains found inorganisms from all domains of life. An intein (intervening protein)carries out a unique auto-processing event known as protein splicing inwhich it excises itself out from a larger precursor polypeptide throughthe cleavage of two peptide bonds and, in the process, ligates theflanking extein (external protein) sequences through the formation of anew peptide bond. This rearrangement occurs post-translationally (orpossibly co-translationally), as intein genes are found embedded inframe within other protein-coding genes. Furthermore, intein-mediatedprotein splicing is spontaneous; it requires no external factor orenergy source, only the folding of the intein domain. This process isalso known as cis-protein splicing, as opposed to the natural process oftrans-protein splicing with “split inteins.” Inteins are the proteinequivalent of the self-splicing RNA introns (see Perler et al., NucleicAcids Res. 22:1125-1127 (1994)), which catalyze their own excision froma precursor protein with the concomitant fusion of the flanking proteinsequences, known as exteins (reviewed in Perler et al., Curr. Opin.Chem. Biol. 1:292-299 (1997); Perler, F. B. Cell 92(1):1-4 (1998); Xu etal., EMBO J. 15(19):5146-5153 (1996)).

The mechanism of the protein splicing process has been studied in greatdetail (Chong, et al., J. Biol. Chem. 1996, 271, 22159-22168; Xu, M-Q &Perler, F. B. EMBO Journal, 1996, 15, 5146-5153) and conserved aminoacids have been found at the intein and extein splicing points (Xu, etal., EMBO Journal, 1994, 13 5517-522).

Inteins can also exist as two fragments encoded by two separatelytranscribed and translated genes. These so-called split inteinsself-associate and catalyze protein-splicing activity in trans. Splitinteins have been identified in diverse cyanobacteria and archaea (Caspiet al, Mol Microbiol. 50: 1569-1577 (2003); Choi J. et al, J Mol Biol.556: 1093-1106 (2006.); Dassa B. et al, Biochemistry. 46:322-330(2007.); Liu X. and Yang J., J Biol Chem. 275:26315-26318 (2003); Wu H.et al. Proc Natl Acad Sci USA. £5:9226-9231 (1998.); and Zettler J. etal, FEBS Letters. 553:909-914 (2009)), but have not been found ineukaryotes thus far. Recently, a bioinformatic analysis of environmentalmetagenomic data revealed 26 different loci with a novel genomicarrangement. At each locus, a conserved enzyme coding region isinterrupted by a split intein, with a freestanding endonuclease geneinserted between the sections coding for intein subdomains. Among them,five loci were completely assembled: DNA helicases (gp41-1, gp41-8);Inosine-5′-monophosphate dehydrogenase (IMPDH-1); and Ribonucleotidereductase catalytic subunits (NrdA-2 and NrdJ-1). This fractured geneorganization appears to be present mainly in phages (Dassa et al,Nucleic Acids Research. 57:2560-2573 (2009)).

In certain embodiments, the prime editors described herein can be usedto insert split-intein tags in two different proteins, causing theirintracellular ligation when co-expressed to form a fusion protein. Inprotein trans-splicing, one precursor protein consists of an N-exteinpart followed by the N-intein, another precursor protein consists of theC-intein followed by a C-extein part, and a trans-splicing reaction(catalyzed by the N- and C-inteins together) excises the two inteinsequences and links the two extein sequences with a peptide bond.Protein trans-splicing, being an enzymatic reaction, can work with verylow (e.g., micromolar) concentrations of proteins and can be carried outunder physiological conditions.

The split intein Npu DnaE was characterized as having the highest ratereported for the protein trans-splicing reaction. In addition, the NpuDnaE protein splicing reaction is considered robust and high-yieldingwith respect to different extein sequences, temperatures from 6 to 37°C., and the presence of up to 6M Urea (Zettler J. et al, FEBS Letters.553:909-914 (2009); Iwai I. et al, FEBS Letters 550: 1853-1858 (2006)).As expected, when the Cysl Ala mutation at the N-domain of these inteinswas introduced, the initial N to S-acyl shift and therefore proteinsplicing was blocked. Unfortunately, the C-terminal cleavage reactionwas also almost completely inhibited. The dependence of the asparaginecyclization at the C-terminal splice junction on the acyl shift at theN-terminal scissile peptide bond seems to be a unique property common tothe naturally split DnaE intein alleles (Zettler J. et al. FEBS Letters.555:909-914 (2009)).

Protein trans-splicing, catalyzed by split inteins, provides an entirelyenzymatic method for protein ligation. A split-intein is essentially acontiguous intein (e.g. a mini-intein) split into two pieces namedN-intein and C-intein, respectively. The N-intein and C-intein of asplit intein can associate non-covalently to form an active intein andcatalyze the splicing reaction essentially in same way as a contiguousintein does. Split inteins have been found in nature and also engineeredin laboratories. As used herein, the term “split intein” refers to anyintein in which one or more peptide bond breaks exists between theN-terminal and C-terminal amino acid sequences such that the N-terminaland C-terminal sequences become separate molecules that cannon-covalently reassociate, or reconstitute, into an intein that isfunctional for trans-splicing reactions. Any catalytically activeintein, or fragment thereof, may be used to derive a split intein foruse in the methods of the invention. For example, in one aspect thesplit intein may be derived from a eukaryotic intein. In another aspect,the split intein may be derived from a bacterial intein. In anotheraspect, the split intein may be derived from an archaeal intein.Preferably, the split intein so-derived will possess only the amino acidsequences essential for catalyzing trans-splicing reactions.

Split inteins may be created from contiguous inteins by engineering oneor more split sites in the unstructured loop or intervening amino acidsequence between the −12 conserved beta-strands found in the structureof mini-inteins. Some flexibility in the position of the split sitewithin regions between the beta-strands may exist, provided thatcreation of the split will not disrupt the structure of the intein, thestructured beta-strands in particular, to a sufficient degree thatprotein splicing activity is lost.

The prime editors described herein may incorporate peptide tags(including inteins) into the C-terminal end of a protein of interest. Inother embodiments, the peptide tags (including inteins) may beincorporated into the N-terminal end of a protein of interest. Thepeptide tags may also be incorporated into the interior of a protein ofinterest. The resulting fusion proteins created by the herein describedprime editors may have the following structures:

-   -   [protein of interest]-[peptide tag];    -   [peptide tag]-[protein of interest]; or    -   [protein of interest-N-terminal region]-[peptide tag]-[protein        of interest-C-terminal region].

The principles of guide RNA design for use in peptide tagging throughoutmay be applied to peptide tagging. For example, in one embodiment, thePEgRNA structure for peptide tagging may have the following structure:5′-[spacer sequence]-[gRNA core or scaffold]-[extension arm]-3′, whereinthe extension arm comprises in the 5′ to 3′ direction, a homology arm,edit template (comprising the sequence that encodes the peptide tag),and a primer binding site. This configuration is depicted in FIG. 3D andin FIG. 24 .

In another embodiment, the PEgRNA structure for peptide tagging may havethe following structure: 5′-[extension arm]-[spacer sequence]-[gRNA coreor scaffold]-3′, wherein the extension arm comprises in the 5′ to 3′direction, a homology arm, edit template (comprising the sequence thatencodes the peptide tag), and a primer binding site. This configurationis depicted in FIG. 3E.

Embodiments of peptide tagging using prime editing is depicted in FIGS.25 and 26 and described in Example 4.

E. Use of Prime Editing for Preventing or Treating Prion Disease

Prime editing can also be used to prevent or halt the progression ofprion disease through the installation of one or more protectivemutations into prion proteins (PRNP) which become misfolded during thecourse of disease. Prion diseases or transmissible spongiformencephalopathies (TSEs) are a family of rare progressiveneurodegenerative disorders that affect both humans and animals. Theyare distinguished by long incubation periods, characteristic spongiformchanges associated with neuronal loss, and a failure to induceinflammatory response.

In humans, prion disease includes Creutzfeldt-Jakob Disease (CJD),Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-ScheinkerSyndrome, Fatal Familial Insomnia, and Kuru. In animals, prion diseaseincludes Bovine Spongiform Encephalopathy (BSE or “mad cow disease”),Chronic Wasting Disease (CWD), Scrapie, Transmissible MinkEncephalopathy, Feline Spongiform Encephalopathy, and UngulateSpongiform Encephalopathy. Prime editing may be used to installprotective point mutations into a prion protein in order to prevent orhalt the progression of any one of these prion diseases.

Classic CJD is a human prion disease. It is a neurodegenerative disorderwith characteristic clinical and diagnostic features. This disease israpidly progressive and always fatal. Infection with this disease leadsto death usually within 1 year of onset of illness. CJD is a rapidlyprogressive, invariably fatal neurodegenerative disorder believed to becaused by an abnormal isoform of a cellular glycoprotein known as theprion protein. CJD occurs worldwide and the estimated annual incidencein many countries, including the United States, has been reported to beabout one case per million population. The vast majority of CJD patientsusually die within 1 year of illness onset. CJD is classified as atransmissible spongiform encephalopathy (TSE) along with other priondiseases that occur in humans and animals. In about 85% of patients, CJDoccurs as a sporadic disease with no recognizable pattern oftransmission. A smaller proportion of patients (5 to 15%) develop CJDbecause of inherited mutations of the prion protein gene. Theseinherited forms include Gerstmann-Straussler-Scheinker syndrome andfatal familial insomnia. No treatment is currently known for CJD.

Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease that wasfirst described in 1996 in the United Kingdom. There is now strongscientific evidence that the agent responsible for the outbreak of priondisease in cows, bovine spongiform encephalopathy (BSE or ‘mad cow’disease), is the same agent responsible for the outbreak of vCJD inhumans. Variant CJD (vCJD) is not the same disease as classic CJD (oftensimply called CJD). It has different clinical and pathologiccharacteristics from classic CJD. Each disease also has a particulargenetic profile of the prion protein gene. Both disorders are invariablyfatal brain diseases with unusually long incubation periods measured inyears, and are caused by an unconventional transmissible agent called aprion. No treatment is currently known for vCJD.

BSE (bovine spongiform encephalopathy or “mad cow disease”) is aprogressive neurological disorder of cattle that results from infectionby an unusual transmissible agent called a prion. The nature of thetransmissible agent is not well understood. Currently, the most acceptedtheory is that the agent is a modified form of a normal protein known asprion protein. For reasons that are not yet understood, the normal prionprotein changes into a pathogenic (harmful) form that then damages thecentral nervous system of cattle. There is increasing evidence thatthere are different strains of BSE: the typical or classic BSE strainresponsible for the outbreak in the United Kingdom and two atypicalstrains (H and L strains). No treatment is currently known for BSE.

Chronic wasting disease (CWD) is a prion disease that affects deer, elk,reindeer, sika deer and moose. It has been found in some areas of NorthAmerica, including Canada and the United States, Norway and South Korea.It may take over a year before an infected animal develops symptoms,which can include drastic weight loss (wasting), stumbling, listlessnessand other neurologic symptoms. CWD can affect animals of all ages andsome infected animals may die without ever developing the disease. CWDis fatal to animals and there are no treatments or vaccines.

The causative agents of TSEs are believed to be prions. The term“prions” refers to abnormal, pathogenic agents that are transmissibleand are able to induce abnormal folding of specific normal cellularproteins called prion proteins that are found most abundantly in thebrain. The functions of these normal prion proteins are still notcompletely understood. The abnormal folding of the prion proteins leadsto brain damage and the characteristic signs and symptoms of thedisease. Prion diseases are usually rapidly progressive and alwaysfatal.

As used herein, the term “prion” shall mean an infectious particle knownto cause diseases (spongiform encephalopathies) in humans and animals.The term “prion” is a contraction of the words “protein” and “infection”and the particles are comprised largely if not exclusively of PRNP^(Sc)molecules encoded by a PRNP gene which expresses PRNP^(C) which changesconformation to become PRNP^(Sc). Prions are distinct from bacteria,viruses and viroids. Known prions include those which infect animals tocause scrapie, a transmissible, degenerative disease of the nervoussystem of sheep and goats as well as bovine spongiform encephalopathies(BSE) or mad cow disease and feline spongiform encephalopathies of cats.Four prion diseases, as discussed above, known to affect humans are (1)kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI). As used herein prion includes all forms of prionscausing all or any of these diseases or others in any animals used—andin particular in humans and in domesticated farm animals.

In general, and without wishing to be bound by theory, prior diseasesare caused by misfolding of prion proteins. Such diseases—often calleddeposition diseases—the misfolding of the prion proteins can beaccounted for as follows. If A is the normally synthesized gene productthat carries out an intended physiologic role in a monomeric oroligomeric state, A* is a conformationally activated form of A that iscompetent to undergo a dramatic conformational change, B is theconformationally altered state that prefers multimeric assemblies (i.e.,the misfolded form which forms depositions) and B. is the multimericmaterial that is pathogenic and relatively difficult to recycle. For theprion diseases, PRNP^(C) and PRNP^(Sc) correspond to states A and B.where A is largely helical and monomeric and B_(n) is β-rich andmultimeric.

It is known that certain mutations in prion proteins can be associatedwith increased risk of prior disease. Conversely, certain mutations inprion proteins can be protective in nature. See Bagynszky et al.,“Characterization of mutations in PRNP (prion) gene and their possibleroles in neurodegenerative diseases,” Neuropsychiatr Dis Treat., 2018;14: 2067-2085, the contents of which are incorporated herein byreference.

PRNP (NCBI RefSeq No. NP_000302.1 (SEQ ID NO: 291))—the human prionprotein—is encoded by a 16 kb long gene, located on chromosome 20(4686151-4701588). It contains two exons, and the exon 2 carries theopen reading frame which encodes the 253 amino acid (AA) long PrPprotein. Exon 1 is a noncoding exon, which may serve as transcriptionalinitiation site. The post-translational modifications result in theremoval of the first 22 AA N-terminal fragment (NTF) and the last 23 AAC-terminal fragment (CTF). The NTF is cleaved after PrP transport to theendoplasmic reticulum (ER), while the CTF (glycosylphosphatidylinositol[GPI] signal peptide [GPI-SP]) is cleaved by the GPI anchor. GPI anchorcould be involved in PrP protein transport. It may also play a role ofattachment of prion protein into the outer surface of cell membrane.Normal PrP is composed of a long N-terminal loop (which contains theoctapeptide repeat region), two short p sheets, three a helices, and aC-terminal region (which contains the GPI anchor). Cleavage of PrPresults in a 208 AA long glycoprotein, anchored in the cell membrane.

The amino acid sequence of PRNP (NP_000302.1) is as follows:

(SEQ ID NO: 291) MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG.

The amino acid sequence of PRNP (NP_000302.1) is encoded by thefollowing nucleotide sequence (NCBI Ref. Seq No. NM_000311.5, “Homosapiens prion protein (PRNP), transcript variant 1, mRNA), is asfollows:

(SEQ ID NO: 292) GCGAACCTTGGCTGCTGGATGCTGGTTCTCTTTGTGGCCACATGGAGTGACCTGGGCCTCTGCAAGAAGCGCCCGAAGCCTGGAGGATGGAACACTGGGGGCAGCCGATACCCGGGGCAGGGCAGCCCTGGAGGCAACCGCTACCCACCTCAGGGCGGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCCCCATGGTGGTGGCTGGGGACAGCCTCATGGTGGTGGCTGGGGTCAAGGAGGTGGCACCCACAGTCAGTGGAACAAGCCGAGTAAGCCAAAAACCAACATGAAGCACATGGCTGGTGCTGCAGCAGCTGGGGCAGTGGTGGGGGGCCTTGGCGGCTACATGCTGGGAAGTGCCATGAGCAGGCCCATCATACATTTCGGCAGTGACTATGAGGACCGTTACTATCGTGAAAACATGCACCGTTACCCCAACCAAGTGTACTACAGGCCCATGGATGAGTACAGCAACCAGAACAACTTTGTGCACGACTGCGTCAATATCACAATCAAGCAGCACACGGTCACCACAACCACCAAGGGGGAGAACTTCACCGAGACCGACGTTAAGATGATGGAGCGCGTGGTTGAGCAGATGTGTATCACCCAGTACGAGAGGGAATCTCAGGCCTATTACCAGAGAGGATCGAGCATGGTCCTCTTCTCCTCTCCACCTGTGATCCTCCTGATCTCTTTCCTCATCTTCCTGATAGTGGGATGAGGAAGGTCTTCCTGTTTTCACCATCTTTCTAATCTTTTTCCAGCTTGAGGGAGGCGGTATCCACCTGCAGCCCTTTTAGTGGTGGTGTCTCACTCTTTCTTCTCTCTTTGTCCCGGATAGGCTAATCAATACCCTTGGCACTGATGGGCACTGGAAAACATAGAGTAGACCTGAGATGCTGGTCAAGCCCCCTTTGATTGAGTTCATCATGAGCCGTTGCTAATGCCAGGCCAGTAAAAGTATAACAGCAAATAACCATTGGTTAATCTGGACTTATTTTTGGACTTAGTGCAACAGGTTGAGGCTAAAACAAATCTCAGAACAGTCTGAAATACCTTTGCCTGGATACCTCTGGCTCCTTCAGCAGCTAGAGCTCAGTATACTAATGCCCTATCTTAGTAGAGATTTCATAGCTATTTAGAGATATTTTCCATTTTAAGAAAACCCGACAACATTTCTGCCAGGTTTGTTAGGAGGCCACATGATACTTATTCAAAAAAATCCTAGAGATTCTTAGCTCTTGGGATGCAGGCTCAGCCCGCTGGAGCATGAGCTCTGTGTGTACCGAGAACTGGGGTGATGTTTTACTTTTCACAGTATGGGCTACACAGCAGCTGTTCAACAAGAGTAAATATTGTCACAACACTGAACCTCTGGCTAGAGGACATATTCACAGTGAACATAACTGTAACATATATGAAAGGCTTCTGGGACTTGAAATCAAATGTTTGGGAATGGTGCCCTTGGAGGCAACCTCCCATTTTAGATGTTTAAAGGACCCTATATGTGGCATTCCTTTCTTTAAACTATAGGTAATTAAGGCAGCTGAAAAGTAAATTGCCTTCTAGACACTGAAGGCAAATCTCCTTTGTCCATTTACCTGGAAACCAGAATGATTTTGACATACAGGAGAGCTGCAGTTGTGAAAGCACCATCATCATAGAGGATGATGTAATTAAAAAATGGTCAGTGTGCAAAGAAAAGAACTGCTTGCATTTCTTTATTTCTGTCTCATAATTGTCAAAAACCAGAATTAGGTCAAGTTCATAGTTTCTGTAATTGGCTTTTGAATCAAAGAATAGGGAGACAATCTAAAAAATATCTTAGGTTGGAGATGACAGAAATATGATTGATTTGAAGTGGAAAAAGAAATTCTGTTAATGTTAATTAAAGTAAAATTATTCCCTGAATTGTTTGATATTGTCACCTAGCAGATATGTATTACTTTTCTGCAATGTTATTATTGGCTTGCACTTTGTGAGTATTCTATGTAAAAATATATATGTATATAAAATATATATTGCATAGGACAGACTTAGGAGTTTTGTTTAGAGCAGTTAACATCTGAAGTGTCTAATGCATTAACTTTTGTAAGGTACTGAATACTTAATATGTGGGAAACCCTTTTGCGTGGTCCTTAGGCTTACAATGTGCACTGAATCGTTTCATGTAAGAATCCAAAGTGGACACCATTAACAGGTCTTTGAAATATGCATGTACTTTATATTTTCTATATTTGTAACTTTGCATGTTCTTGTTTTGTTATATAAAAAAATTGTAAATGTTTAATATCTGACTGAAATTAAA CGAGCGAAGATGAGCACCA

Mutation sites relative to PRNP (NP_000302.1) which are linked to CJDand FFI are reported are as follows. These mutations can be removed orinstalled using the prime editors disclosed herein.

AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO CJD PRIONDISEASE (SEE TABLE 1 OF BAGYNSZKY ET AL., 2018) MUTATION (RELATIVE TO SEQ ID NO: 291 OF PRNP NP_000302.1) D178NMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHNCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 293) T188KMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHKVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 294) E196KMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGKNFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 295) E196AMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGANFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 296)E200K MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTKTDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 297) E200GMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTGTDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 298)V203I MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDIKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 299) R208HMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMEHVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 300) V210IMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVIEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 301) E211QMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVQQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 302) M232RMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSRVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 303)

Mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) which arelinked to GSS are reported, as follows:

AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO GSS PRIONDISEASE (SEE TABLE 2 OF BAGYNSZKY ET AL., 2018) MUTATION(RELATIVE TO SEQ ID NO: 291 OF PRNP NP_000302.1) P102LMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKLSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 304) P105LMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKLKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 305) A117VMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAVAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 306) G131VMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLVSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 307) V176GMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFGHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 308) H187RMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQRTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 309)MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 291) F198SMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENSTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 311) D202NMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETNVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 312) Q212PMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEPMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 313) Q217RMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITRYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 314) M232TMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSTVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 315)

Mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) which arelinked to a possible protective nature against prion disease, asfollows:

AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO A PROTECTIVENATURE AGAINST PRION DISEASE (SEE TABLE 4 OF BAGYNSZKY ET MUTATIONAL., 2018) (RELATIVE TO SEQ ID NO: 291 OF PRNP NP_000302.1) G127SMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGSYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 316) G127VMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGVYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 317) M129VMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYVLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 318) D167GMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMGEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 319) D167NMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMNEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 320) N171SMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSSQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 321) E219KMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYKRESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 322) P238SMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSSPVILLISFLIFLIVG (SEQ ID NO: 323)

Thus, in various embodiments, prime editing may be used to remove amutation in PRNP that is linked to prion disease or install a mutationin PRNP that is considered to be protective against prion disease. Forexample, prime editing may be use to remove or restore a D178N, V180I,T188K, E196K, E196A, E200K, E200G, V203I, R208H, V210I, E211Q, I215V, orM232R mutation in the PRNP protein (relative to PRNP of NP_000302.1)(SEQ ID NO: 291). In other embodiments, prime editing may be use toremove or restore a P102L, P105L, A117V, G131V, V176G, H187R, F198S,D202N, Q212P, Q217R, or M232T mutation in the PRNP protein (relative toPRNP of NP_000302.1) (SEQ ID NO: 291). By removing or correcting for thepresence of such mutations in PRNP using prime editing, the risk ofprion disease may be reduced or eliminated.

In other embodiments, prime editing may be used to install a protectivemutation in PRNP that is linked to a protective effect against one ormore prion diseases. For example, prime editing may be used to install aG127S, G127V, M129V, D167G, D167N, N171S, E219K, or P238S protectivemutation in PRNP (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). Instill other embodiments, the protective mutation may be any alternateamino acid installed at G127, G127, M129, D167, D167, N171, E219, orP238 in PRNP (relative to PRNP of NP_000302.1) (SEQ ID NO: 291).

In particular embodiments, prime editing may be used to install a G127Vprotective mutation in PRNP, as illustrated in FIG. 27 and discussed inExample 5.

In another embodiment, prime editing may be used to install an E219Kprotective mutation in PRNP.

The PRNP protein and the protective mutation site are conserved inmammals, so in addition to treating human disease it could also be usedto generate cows and sheep that are immune to prion disease, or evenhelp cure wild populations of animals that are suffering from priondisease. Prime editing has already been used to achieve ˜25%installation of a naturally occurring protective allele in human cells,and previous mouse experiments indicate that this level of installationis sufficient to cause immunity from most prion diseases. This method isthe first and potentially only current way to install this allele withsuch high efficiency in most cell types. Another possible strategy fortreatment is to use prime editing to reduce or eliminate the expressionof PRNP by installing an early stop codon in the gene.

Using the principles described herein for PEgRNA design, appropriatePEgRNAs may be designed for installing desired protective mutations, orfor removing prion disease-associated mutations from PRNP. For example,the below list of PEgRNAs can be used to install the G127V protectiveallele and the E219K protective allele in human PRNP, as well as theG127V protective allele in PRNP of various animals.

HUMAN PEGRNA FOR GCAGTGGTGGGGGGCCTTGGGTTTTAGAGCTAGAAINSTALLATION OF G127V ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGIN HUMAN PRNP: AAAAAGTGGCACCGAGTCGGTGCATGTAGACGCCA

 (SEQ ID NO: 324) HUMAN PEGRNA FOR TGTGTATCACCCAGTACGAGGTTTTAGAGCTAGAAAINSTALLATION OF E219K TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAIN HUMAN PRNP AAAAGTGGCACCGAGTCGGTGC AGATTCTCTCTT

 (SEQ ID NO: 325) COW (BOS TAURUS) GCAGTGGTAGGGGGCCTTGGGTTTTAGAGCTAGAAPEGRNA FOR ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG INSTALLATION OF G127VAAAAAGTGGCACCGAGTCGGTGCATGTAGACACCA

IN COW PRNP

 (SEQ ID NO: 326) HAMSTER GCCGTGGTGGGGGGCCTTGGGTTTTAGAGCTAGAA(MESOCRICETUS ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AURATUS) PEGRNA FORAAAAAGTGGCACCGAGTCGGTGCATGTAGACACCA

INSTALLATION OF G127V

 (SEQ ID NO: 327) IN HAMSTER PRNP MOUSE (MUSGCAGTAGTGGGGGGCCTTGGGTTTTAGAGCTAGAA MUSCULUS) PEGRNAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG FOR INSTALLATION OFAAAAAGTGGCACCGAGTCGGTGCATGTAGACACCA

G127V IN MOUST PRNP

 (SEQ ID NO: 328) DEER (ODOCOILEUS GCAGTGGTAGGGGGCCTTGGGTTTTAGAGCTAGAAVIRGINIANUS) PEGRNA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGFOR INSTALLATION OF AAAAAGTGGCACCGAGTCGGTGCATGTAGACACCA

G127V IN DEER PNRP

 (SEQ ID NO: 329) FERRET (MUSTELA GCGGTTGTGGGGGGCCTGGGGTTTTAGAGCTAGAAPUTORIUS FURO) ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG PEGRNA FORAAAAAGTGGCACCGAGTCGGTGCATGTAGACGCCC

INSTALLATION OF G127V

 (SEQ ID NO: 330) IN FERRET PRNP KEY: SPACER IS BOLDED. SGRNA SCAFFOLDIS NORMAL TEXT. RT TEMPLATE IS ONCE UNDERLINED. PBS IS TWICE UNDERLINED.

F. Use of Prime Editing for RNA Tagging

Prime editing may also be used to manipulate, alter, and otherwisemodify the sequences of DNA encoding RNA functions through RNA tagging,and in this way provides a means to indirectly modify the structure andfunction of RNA. For example, PE can be used to insert motifs that arefunctional at the RNA level (hereafter RNA motifs) to tag or otherwisemanipulate non-coding RNAs or mRNAs. These motifs could serve toincrease gene expression, decrease gene expression, alter splicing,change post-transcriptional modification, affect the sub-cellularlocation of the RNA, enable isolation or determination of the intra- orextra-cellular location of the RNA (using, for instance, fluorescent RNAaptamers such as Spinach, Spinach2, Baby Spinach, or Broccoli), recruitendogenous or exogenous protein or RNA binders, introduce sgRNAs, orinduce processing of the RNA, by either self-cleavage or RNAses (seeFIG. 28B and Example 6 for further details).

The following RNA tags or motifs may be inserted into a gene of interestusing prime editing with an appropriate PEgRNA (designed using theguidance provided herein) to affect various properties of RNA, includingRNA transport, expression level, splicing, and detection.

EXEMPLARY PEGRNA FOR PRIME EDITING INSERTION OF RNA MOTIF INTO FUNCTION/ THE EXEMPLARY RNA MOTIF SEQUENCE OF RNA MOTIF EFFECTHEXA GENE* POLYOMAVIRUS AACTTGTTTATTGCAGCTT TERMINATION ATCCTTCCAGTCAGSIMIAN ATAATGGTTACAAATAAAG OF GGCCATGTTTGAGA VIRUS 40CAATAGCATCACAAATTTC TRANSCRIPTION GCTAGAAATAGCAA (SV40) TYPE1ACAAATAAAGCATTTTTTT OF THE GTTTAAATAAGGCT CACTGCATTCTAGTTGTGG TAGGEDAGTCCGTTATCAAC TTTGTCCAAACTCATCAAT GENE; TTGAAAAAGTGGGGTATCTTA (SEQ ID NO: TRANSPORT ACCGAGTCGGTCC A 331) OF MRNACCTGAACCGTATATC INTO TAAGATACATTGAT CYTOSOL; GAGTTTGGACAAA INCREASEDCCACAACTAGAAT RNA GCAGTGAAAAAAA STABILITY TGCTTTATTTGTG ANDAAATTTGTGATGC EXPRESSION TATTGCTTTATTTG OF ENCODED TAACCATTATAAGCPROTEIN TGCAATAAACAAG TTCTATGGCCCTGA CTGGAA (SEQ ID NO: 332)POLYOMAVIRUS CCATGGCCCAACTTGTTTA TERMINATION ATCCTTCCAGTCAG SIMIANTTGCAGCTTATAATGGTTA OF GGCCATGTTTGAGA VIRUS 40 CAAATAAAGCAATAGCATTRANSCRIPTION GCTAGAAATAGCAA (SV40) TYPE2 CACAAATTTCACAAATAA OF THEGTTTAAATAAGGCT AGCATTTTTTTCACTGCAT TAGGED AGTCCGTTATCAACTCTAGTTGTGGTTTGTCCA GENE; TTGAAAAAGTGGG AACTCATCAATGTATCTTA TRANSPORTACCGAGTCGGTCC A TCATGTCTGGATCTC (SEQ OF MRNA CCTGAACCGTATATC ID NO: 333)INTO GAGATCCAGACAT CYTOSOL; GATAAGATACATT INCREASED GATGAGTTTGGAC RNAAAACCACAACTAG STABILITY AATGCAGTGAAAA AND AAATGCTTTATTT EXPRESSIONGTGAAATTTGTGA OF ENCODED TGCTATTGCTTTAT PROTEIN TTGTAACCATTATAAGCTGCAATAAAC AAGTTGGGCCATG GCTATGGCCCTGAC TGGAA (SEQ ID NO: 334)POLYOMAVIRUS TGATCATAATCAAGCCATA TERMINATION ATCCTTCCAGTCAG SIMIANTCACATCTGTAGAGGTTTA OF GGCCATGTTTGAGA VIRUS 40 CTTGCTTTAAAAAACCTCTRANSCRIPTION GCTAGAAATAGCAA (SV40) TYPE3 CACACCTCCCCCTGAACC OF THEGTTTAAATAAGGCT TGAAACATAAAATGAATG TAGGED AGTCCGTTATCAACCAATTGTTGTTGTTAACTT GENE; TTGAAAAAGTGGG GTTTATTGCAGCTTATAAT TRANSPORTACCGAGTCGGTCC A GGTTACAAATAAAGCAAT OF MRNA CCTGAACCGTATATCAGCATCACAAATTTCACA INTO GCAGATCCAGACA AATAAAGCATTTTTTTCAC CYTOSOL;TGATAAGATACATT TGCATTCTAGTTGTGGTTT INCREASED GATGAGTTTGGACGTCCAAACTCATCAATGTA RNA AAACCACAACTAG TCTTATCATGTCTGGATCT STABILITYAATGCAGTGAAAA GC (SEQ ID NO: 335) AND AAATGCTTTATTT EXPRESSIONGTGAAATTTGTGA OF ENCODED TGCTATTGCTTTAT PROTEIN TTGTAACCATTATAAGCTGCAATAAAC AAGTTAACAACAA CAATTGCATTCAT TTTATGTTTCAGG TTCAGGGGGAGGTGTGGAGGTTTTT TAAAGCAAGTAAA CCTCTACAGATGT GATATGGCTTGAT TATGATCACTATGGCCCTGACTGGAA (SEQ ID NO: 336) HUMAN ACGGGTGGCATCCCTGTG TRANSPORTATCCTTCCAGTCAG GROWTH ACCCCTCCCCAGTGCCTC OF RNA INTO GGCCATGTTTGAGAHORMONE TCCTGGCCCTGGAAGTTG CYTOPLASM; GCTAGAAATAGCAA (HGH)CCACTCCAGTGCCCACCA ENHANCED GTTTAAATAAGGCT GCCTTGTCCTAATAAAATT RNAAGTCCGTTATCAAC AAGTTGCATCATTTTGTCT STABILITY TTGAAAAAGTGGGGACTAGGTGTCCTTCTATA AND ACCGAGTCGGTCC A ATATTATGGGGTGGAGGG EXPRESSIONCCTGAACCGTATATC GGGTGGTATGGAGCAAGG OF ENCODED AAGGACAGGGAAGGCAAGTTGGGAAGACA PROTEIN GGGAGCAGTGGT ACCTGTAGGGCCTGCGGG TCACGCCTGTAATGTCTATTGGGAACCAAGC CCCAGCAATTTGG TGGAGTGCAGTGGCACAA GAGGCCAAGGTGTCTTGGCTCACTGCAATCT GGTAGATCACCTG CCGCCTCCTGGGTTCAAG AGATTAGGAGTTGCGATTCTCCTGCCTCAGCC GAGACCAGCCTG TCCCGAGTTGTTGGGATT GCCAATATGGTGACCAGGCATGCATGACCAG AACCCCGTCTCTA GCTCAGCTAATTTTTGTTT CCAAAAAAACAAATTTTGGTAGAGACGGGGT AATTAGCTGAGCC TTCACCATATTGGCCAGGC TGGTCATGCATGCTGGTCTCCAACTCCTAATC CTGGAATCCCAAC TCAGGTGATCTACCCACCT AACTCGGGAGGCTTGGCCTCCCAAATTGCTG GAGGCAGGAGAAT GGATTACAGGCGTGAACC CGCTTGAACCCAGACTGCTCCCTTCCCTGTCC GAGGCGGAGATTG TT (SEQ ID NO: 337) CAGTGAGCCAAGATTGTGCCACTGCA CTCCAGCTTGGTT CCCAATAGACCCC GCAGGCCCTACAG GTTGTCTTCCCAACTTGCCCCTTGCT CCATACCACCCCC CTCCACCCCATAA TATTATAGAAGGA CACCTAGTCAGACAAAATGATGCAAC TTAATTTTATTAGG ACAAGGCTGGTG GGCACTGGAGTG GCAACTTCCAGGGCCAGGAGAGGCA CTGGGGAGGGGT CACAGGGATGCCA CCCGTCTATGGCCC TGACTGGAA(SEQ ID NO: 338) BOVINE CGACTGTGCCTTCTAGTTG TRANSPORT ATCCTTCCAGTCAGGROWTH CCAGCCATCTGTTGTTTGC OF RNA INTO GGCCATGTTTGAGA HORMONECCCTCCCCCGTGCCTTCCT CYTOPLASM; GCTAGAAATAGCAA (BGH) TGACCCTGGAAGGTGCCAENHANCED GTTTAAATAAGGCT CTCCCACTGTCCTTTCCTA RNA AGTCCGTTATCAACATAAAATGAGGAAATTGC STABILITY TTGAAAAAGTGGG ATCGCATTGTCTGAGTAGG ANDACCGAGTCGGTCC A TGTCATTCTATTCTGGGGG EXPRESSION CCTGAACCGTATATCGTGGGGTGGGGCAGGAC OF ENCODED CCATAGAGCCCAC AGCAAGGGGGAGGATTG PROTEINCGCATCCCCAGCA GGAAGACAATAGCAGGCA TGCCTGCTATTGT TGCTGGGGATGCGGTGGGCTTCCCAATCCTC CTCTATGG (SEQ ID NO: CCCCTTGCTGTCC 339) TGCCCCACCCCACCCCCCAGAATAGA ATGACACCTACTC AGACAATGCGATG CAATTTCCTCATTT TATTAGGAAAGGACAGTGGGAGTGG CACCTTCCAGGGT CAAGGAAGGCAC GGGGGAGGGGCA AACAACAGATGGCTGGCAACTAGAAG GCACAGTCGCTAT GGCCCTGACTGGAA (SEQ ID NO: 340) RABBITTTCACTCCTCAGGTGCAG TRANSPORT ATCCTTCCAGTCAG BETA- GCTGCCTATCAGAAGGTGOF RNA INTO GGCCATGTTTGAGA GLOBIN GTGGCTGGTGTGGCCAAT CYTOPLASM;GCTAGAAATAGCAA (RBGLOB) GCCCTGGCTCACAAATAC ENHANCED GTTTAAATAAGGCTCACTGAGATCTTTTTCCCT RNA AGTCCGTTATCAAC CTGCCAAAAATTATGGGG STABILITYTTGAAAAAGTGGG ACATCATGAAGCCCCTTG AND ACCGAGTCGGTCC A AGCATCTGACTTCTGGCTAEXPRESSION CCTGAACCGTATATC ATAAAGGAAATTTATTTTC OF ENCODED GATCTCCATAAGAATTGCAATAGTGTGTTGGA PROTEIN GAAGAGGGACAG ATTTTTTGTGTCTCTCACTCTATGACTGGGAG CGGAAGGACATATGGGAG TAGTCAGGAGAGG GGCAAATCATTTAAAACATAGGAAAAATCTGG CAGAATGAGTATTTGGTTT CTAGTAAAACATG AGAGTTTGGCAACATATGTAAGGAAAATTTT CCCATATGCTGGCTGCCAT AGGGATGTTAAAG GAACAAAGGTTGGCTATAAAAAAAATAACAC AAGAGGTCATCAGTATATG AAAACAAAATATA AAACAGCCCCCTGCTGTCAAAAAAATCTAAC CATTCCTTATTCCATAGAA CTCAAGTCAAGGC AAGCCTTGACTTGAGGTTTTTTCTATGGAATA AGATTTTTTTTATATTTTGT AGGAATGGACAGC TTTGTGTTATTTTTTTCTTTAGGGGGCTGTTTC AACATCCCTAAAATTTTCC ATATACTGATGAC TTACATGTTTTACTAGCCACTCTTTATAGCCA GATTTTTCCTCCTCTCCTG ACCTTTGTTCATG ACTACTCCCAGTCATAGCTGCAGCCAGCATAT GTCCCTCTTCTCTTATGGA GGGCATATGTTGC GATC (SEQ ID NO: 341)CAAACTCTAAACC AAATACTCATTCT GATGTTTTAAATG ATTTGCCCTCCCA TATGTCCTTCCGAGTGAGAGACACAA AAAATTCCAACAC ACTATTGCAATGA AAATAAATTTCCTT TATTAGCCAGAAGTCAGATGCTCAAG GGGCTTCATGATG TCCCCATAATTTTT GGCAGAGGGAAA AAGATCTCAGTGGTATTTGTGAGCCA GGGCATTGGCCAC ACCAGCCACCACC TTCTGATAGGCAG CCTGCACCTGAGGAGTGAACTATGGCC CTGACTGGAA (SEQ ID NO: 342) THYMIDINE GGGGGAGGCTAACTGAAATRANSPORT ATCCTTCCAGTCAG KINASE (TK) CACGGAAGGAGACAATAC OF RNA INTOGGCCATGTTTGAGA CGGAAGGAACCCGCGCTA CYTOPLASM; GCTAGAAATAGCAATGACGGCAATAAAAAGAC ENHANCED GTTTAAATAAGGCT AGAATAAAACGCACGGGT RNAAGTCCGTTATCAAC GTTGGGTCGTTTGTTCATA STABILITY TTGAAAAAGTGGGAACGCGGGGTTCGGTCCC AND ACCGAGTCGGTCC A AGGGCTGGCACTCTGTCG EXPRESSIONCCTGAACCGTATATC ATACCCCACCGAGACCCC OF ENCODED CTATGGCAGGGCCATTGGGGCCAATACGCCC PROTEIN TGCCGCCCCGACG GCGTTTCTTCCTTTTCCCCTTGGCTGCGAGCC ACCCCACCCCCCAAGTTC CTGGGCCTTCACC GGGTGAAGGCCCAGGGCTCGAACTTGGGGG CGCAGCCAACGTCGGGGC GTGGGGTGGGGA GGCAGGCCCTGCCATAGAAAGGAAGAAAC (SEQ ID NO: 343) GCGGGCGTATTGG CCCCAATGGGGTC TCGGTGGGGTATCGACAGAGTGCCA GCCCTGGGACCG AACCCCGCGTTTA TGAACAAACGACC CAACACCCGTGCGTTTTATTCTGTCTT TTTATTGCCGTCA TAGCGCGGGTTCC TTCCGGTATTGTC TCCTTCCGTGTTTCAGTTAGCCTCCC CCCTATGGCCCTGA CTGGAA (SEQ ID NO: 344) MALAT1 ENE-TAGGGTCATGAAGGTTTTT RESULTS IN ATCCTTCCAGTCAG MASCRNA CTTTTCCTGAGAAAACAARETENTION GGCCATGTTTGAGA CACGTATTGTTTTCTCAGG OF RNA IN GCTAGAAATAGCAATTTTGCTTTTTGGCCTTTT NUCLEUS, GTTTAAATAAGGCT TCTAGCTTAAAAAAAAAATRANSCRIPT AGTCCGTTATCAAC AAAGCAAAAGATGCTGGT TERMINATION TTGAAAAAGTGGGGGTTGGCACTCCTGGTTT AND ACCGAGTCGGTCC A CCAGGACGGGGTTCAAAT STABILIZATIONCCTGAACCGTATATC CCCTGCGGCGTCTTTGCTT AGTCAAAGCAAAG TGACT (SEQ ID NO: 345)ACGCCGCAGGGAT TTGAACCCCGTCC TGGAAACCAGGA GTGCCAACCACCA GCATCTTTTGCTTTTTTTTTTTTTAAG CTAGAAAAAGGCC AAAAAGCAAAACC TGAGAAAACAATA CGTGTTGTTTTCTCAGGAAAAGAAAA ACCTTCATGACCC TACTATGGCCCTGA CTGGAA (SEQ ID NO: 346)KSHV PAN TGTTTTGGCTGGGTTTTTC RESULTS IN ATCCTTCCAGTCAG ENECTTGTTCGCACCGGACAC RETENTION GGCCATGTTTGAGA CTCCAGTGACCAGACGGC OF RNA INGCTAGAAATAGCAA AAGGTTTTTATCCCAGTGT NUCLEUS, GTTTAAATAAGGCTATATTGGAAAAACATGTTA TRANSCRIPT AGTCCGTTATCAAC TACTTTTGACAATTTAACGTERMINATION TTGAAAAAGTGGG TGCCTAGAGCTCAAATTA AND ACCGAGTCGGTCC AAACTAATACCATAACGTAA STABILIZATION CCTGAACCGTATATC TGCAACTTACAACATAAATTTTTTTTTTTTTTT AAAGGTCAATGTTTAATCC TTTTTATGGATTAA ATAAAAAAAAAAAAAAAACATTGACCTTTA AAAA (SEQ ID NO: 347) TTTATGTTGTAAG TTGCATTACGTTATGGTATTAGTTTAAT TTGAGCTCTAGGC ACGTTAAATTGTC AAAAGTATAACAT GTTTTTCCAATATACACTGGGATAAAA ACCTTGCCGTCTG GTCACTGGAGGTG TCCGGTGCGAACA AGGAAAAACCCAGCCAAAACACTATG GCCCTGACTGGAA (SEQ ID NO: 348) THREE, TGTTTTGGCTGGGTTTTTCRESULTS IN ATCCTTCCAGTCAG SEQUENTIAL CTTGTTCGCACCGGACAC RETENTIONGGCCATGTTTGAGA KSHV PAN CTCCAGTGACCAGACGGC OF RNA IN GCTAGAAATAGCAAENES WITH AAGGTTTTTATCCCAGTGT NUCLEUS, GTTTAAATAAGGCT SHORT,ATATTGGAAAAACATGTTA TRANSCRIPT AGTCCGTTATCAAC UNCONSERVEDTACTTTTGACAATTTAACG TERMINATION TTGAAAAAGTGGG RNA TGCCTAGAGCTCAAATTA ANDACCGAGTCGGTCC A LINKERS AACTAATACCATAACGTAA STABILIZATION,CCTGAACCGTATATC TGCAACTTACAACATAAAT PREDICTED TTTTTTTTTTTTTTAAAGGTCAATGTTTAATCC TO BE TTTTTATGGATTAA ATAAAAAAAAAAAAAAA GREATERACATTGACCTTTA AAAAACACACTGTTTTGG THAN A TTTATGTTGTAAGCTGGGTTTTTCCTTGTTCG SINGLE PAN TTGCATTACGTTAT CACCGGACACCTCCAGTG ENEGGTATTAGTTTAAT ACCAGACGGCAAGGTTTT TTGAGCTCTAGGC TATCCCAGTGTATATTGGAACGTTAAATTGTC AAAACATGTTATACTTTTG AAAAGTATAACAT ACAATTTAACGTGCCTAGGTTTTTCCAATATA AGCTCAAATTAAACTAATA CACTGGGATAAAA CCATAACGTAATGCAACTTACCTTGCCGTCTG ACAACATAAATAAAGGTC GTCACTGGAGGTG AATGTTTAATCCATAAAAATCCGGTGCGAACA AAAAAAAAAAAAAATCTC AGGAAAAACCCAG TCTGTTTTGGCTGGGTTTTCCAAAACAGAGAG TCCTTGTTCGCACCGGAC ATTTTTTTTTTTTT ACCTCCAGTGACCAGACGTTTTTTATGGATTA GCAAGGTTTTTATCCCAGT AACATTGACCTTT GTATATTGGAAAAACATGTATTTATGTTGTAAG TATACTTTTGACAATTTAA TTGCATTACGTTAT CGTGCCTAGAGCTCAAATGGTATTAGTTTAAT TAAACTAATACCATAACGT TTGAGCTCTAGGC AATGCAACTTACAACATAACGTTAAATTGTC AATAAAGGTCAATGTTTA AAAAGTATAACAT ATCCATAAAAAAAAAAAAGTTTTTCCAATATA AAAAAAA (SEQ ID NO: CACTGGGATAAAA 349) ACCTTGCCGTCTGGTCACTGGAGGTG TCCGGTGCGAACA AGGAAAAACCCAG CCAAAACAGTGTG TTTTTTTTTTTTTTTTTTTTATGGATTA AACATTGACCTTT ATTTATGTTGTAAG TTGCATTACGTTATGGTATTAGTTTAAT TTGAGCTCTAGGC ACGTTAAATTGTC AAAAGTATAACAT GTTTTTCCAATATACACTGGGATAAAA ACCTTGCCGTCTG GTCACTGGAGGTG TCCGGTGCGAACA AGGAAAAACCCAGCCAAAACACTATG GCCCTGACTGGAA (SEQ ID NO: 350) SMBOX/U1 CAGCAAGTTCAGAGAAATRESULTS IN ATCCTTCCAGTCAG SNRNA BOX CTGAACTTGCTGGATTTTT RETENTIONGGCCATGTTTGAGA GGAGCAGGGAGATGGAAT OF RNA IN GCTAGAAATAGCAAAGGAGCTTGCTCCGTCCA NUCLEUS GTTTAAATAAGGCT CTCCACGCATCGACCTGG ANDAGTCCGTTATCAAC TATTGCAGTACCTCCAGG TRANSCRIPT TTGAAAAAGTGGGAACGGTGCACCCACTTTC TERMINATION ACCGAGTCGGTCC A TGGAGTTTCAAAAGTAGACCTGAACCGTATATC CTGTACGCTAAGGGTCATA TTTAAGACGCCAA TCTTTTTTTGTTTGGTTTGCCAAGACACAAAC TGTCTTGGTTGGCGTCTTA CAAACAAAAAAAG AA (SEQ ID NO: 351)ATATGACCCTTAG CGTACAGTCTACT TTTGAAACTCCAG AAAGTGGGTGCAC CGTTCCTGGAGGTACTGCAATACCAG GTCGATGCGTGGA GTGGACGGAGCA AGCTCCTATTCCA TCTCCCTGCTCCAAAAATCCAGCAAG TTCAGATTTCTCT GAACTTGCTGCTA TGGCCCTGACTGGA A(SEQ ID NO: 352) U1 SNRNA 3′ GTTTCAAAAGTAGACTGT RESULTS INATCCTTCCAGTCAG BOX ACGCTAAGGGTCATATCTT RETENTION GGCCATGTTTGAGATTTTTGTTTGGTTTGTGTC OF RNA IN GCTAGAAATAGCAA TTGGTTGGCGTCTTAAA NUCLEUSGTTTAAATAAGGCT (SEQ ID NO: 353) AND AGTCCGTTATCAAC TRANSCRIPTTTGAAAAAGTGGG TERMINATION ACCGAGTCGGTCC A CCTGAACCGTATATC TTTAAGACGCCAACCAAGACACAAAC CAAACAAAAAAAG ATATGACCCTTAG CGTACAGTCTACT TTTGAAACCTATGGCCCTGACTGGAA (SEQ ID NO: 354) TRNA-LYSINE GCCCGGCTAGCTCAGTCG REPORTEDATCCTTCCAGTCAG GTAGAGCATGAGACTCTT TO ENABLE GGCCATGTTTGAGAAATCTCAGGGTCGTGGGT TRANSPORT GCTAGAAATAGCAA TCGAGCCCCACGTTGGGC OF RNA TOGTTTAAATAAGGCT G (SEQ ID NO: 355) MITOCHONDRIA AGTCCGTTATCAACTTGAAAAAGTGGG ACCGAGTCGGTCC A CCTGAACCGTATATC CGCCCAACGTGG GGCTCGAACCCACGACCCTGAGATTA AGAGTCTCATGCT CTACCGACTGAGC TAGCCGGGCCTAT GGCCCTGACTGGAA(SEQ ID NO: 356) BROCCOLI GAGACGGTCGGGTCCAGA VISUALIZATIONATCCTTCCAGTCAG APTAMER TATTCGTATCTGTCGAGTA (FLUORESCENCE) GGCCATGTTTGAGAGAGTGTGGGCTC (SEQ ID GCTAGAAATAGCAA NO: 357) GTTTAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGG ACCGAGTCGGTCC A CCTGAACCGTATATCGAGCCCACACTCT ACTCGACAGATAC GAATATCTGGACC CGACCGTCTCCTA TGGCCCTGACTGGA A(SEQ ID NO: 358) SPINACH GACGCAACTGAATGAAAT VISUALIZATION ATCCTTCCAGTCAGAPTAMER GGTGAAGGACGGGTCCA (FLUORESCENCE) GGCCATGTTTGAGAGGTGTGGCTGCTTCGGCA GCTAGAAATAGCAA GTGCAGCTTGTTGAGTAG GTTTAAATAAGGCTAGTGTGAGCTCCGTAACT AGTCCGTTATCAAC AGTCGCGTC (SEQ ID NO: TTGAAAAAGTGGG359) ACCGAGTCGGTCC A CCTGAACCGTATATC GACGCGACTAGTT ACGGAGCTCACACTCTACTCAACAAG CTGCACTGCCGAA GCAGCCACACCTG GACCCGTCCTTCA CCATTTCATTCAGTTGCGTCCTATGGC CCTGACTGGAA (SEQ ID NO: 360) SPINACH2 GATGTAACTGAATGAAATVISUALIZATION ATCCTTCCAGTCAG APTAMER GGTGAAGGACGGGTCCA (FLUORESCENCE)GGCCATGTTTGAGA GTAGGCTGCTTCGGCAGC GCTAGAAATAGCAA CTACTTGTTGAGTAGAGTGTTTAAATAAGGCT GTGAGCTCCGTAACTAGT AGTCCGTTATCAAC TACATC (SEQ ID NO: 361)TTGAAAAAGTGGG ACCGAGTCGGTCC A CCTGAACCGTATATC GATGTAACTAGTTACGGAGCTCACAC TCTACTCAACAAG TAGGCTGCCGAAG CAGCCTACTGGAC CCGTCCTTCACCATTTCATTCAGTTA CATCCTATGGCCCT GACTGGAA (SEQ ID NO: 362) MANGOGGCACGTACGAAGGGACG VISUALIZATION ATCCTTCCAGTCAG APTAMERGTGCGGAGAGGAGAGTAC (FLUORESCENCE) GGCCATGTTTGAGA GTGC (SEQ ID NO: 363)GCTAGAAATAGCAA GTTTAAATAAGGCT AGTCCGTTATCAAC TTGAAAAAGTGGG ACCGAGTCGGTCCA CCTGAACCGTATATC GCACGTACTCTCC TCTCCGCACCGTC CCTTCGTACGTGCCCTATGGCCCTGAC TGGAA (SEQ ID NO: 364) HDV GGCCGGCATGGTCCCAGC 3′ END RNAATCCTTCCAGTCAG RIBOZYME CTCCTCGCTGGCGCCGGC PROCESSING GGCCATGTTTGAGATGGGCAACATGCTTCGGC GCTAGAAATAGCAA ATGGCGAATGGGAC (SEQ GTTTAAATAAGGCTID NO: 365) AGTCCGTTATCAAC TTGAAAAAGTGGG ACCGAGTCGGTCC A CCTGAACCGTATATCGTCCCATTCGCCA TGCCGAAGCATGT TGCCCAGCCGGC GCCAGCGAGGAG GCTGGGACCATGCCGGCCCTATGGCCC TGACTGGAA (SEQ ID NO: 366) N⁶- GGACTCTAGGACTGGACTTARGET FOR ATCCTTCCAGTCAG METHYLADE TTGGACT (SEQ ID NO: 367) METHYLATIONGGCCATGTTTGAGA NOSINE (UNDERLINED GCTAGAAATAGCAA MARKER A'S AREGTTTAAATAAGGCT (M⁶A) METHYLATED). AGTCCGTTATCAAC M6A TTGAAAAAGTGGGMETHYLATION ACCGAGTCGGTCC A CAN CCTGAACCGTATATC RESULT IN AGTCCAAAGTCCAENHANCED GTCCTAGAGTCCC RNA TATGGCCCTGACTG STABILITY GAA AND(SEQ ID NO: 368) EXPRESSION, BUT IS NOT YET FULLY UNDERSTOOD *eachPEgRNA is shown in the 5′ to 3′ direction and has the followingstructural elements of FIG. 3F as designated by font type, as follows:5′ - spacer sequence (normal font) - gRNA core (underlined sequence) -homology arm (italicized) - RT template (bolded font) - primer bindingsite (italicized) - 3′.

The PEgRNAs of the above table are designed to site-specifically insertexamples of the above motifs into the HEXA gene (defective in Tay-Sachsdisease) (e.g., GenBank No. KR710351.1 (SEQ ID NO: 369), however, thisis only for purposes of illustration. The use of prime editing in RNAtagging is not limited to the HEXA gene and indeed may be any. The HEXAmRNA has the following nucleotide sequence:

(SEQ ID NO: 369) GTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGTTGGCATGACAAGTTCCAGGCTTTGGTTTTCGCTGCTGCTGGCGGCAGCGTTCGCAGGACGGGCGACGGCCCTCTGGCCCTGGCCTCAGAACTTCCAAACCTCCGACCAGCGCTACGTCCTTTACCCGAACAACTTTCAATTCCAGTACGATGTCAGCTCGGCCGCGCAGCCCGGCTGCTCAGTCCTCGACGAGGCCTTCCAGCGCTATCGTGACCTGCTTTTCGGTTCCGGGTCTTGGCCCCGTCCTTACCTCACAGGGAAACGGCATACACTGGAGAAGAATGTGTTGGTTGTCTCTGTAGTCACACCTGGATGTAACCAGCTTCCTACTTTGGAGTCAGTGGAGAATTATACCCTGACCATAAATGATGACCAGTGTTTACTCCTCTCTGAGACTGTCTGGGGAGCTCTCCGAGGTCTGGAGACTTTTAGCCAGCTTGTTTGGAAATCTGCTGAGGGCACATTCTTTATCAACAAGACTGAGATTGAGGACTTTCCCCGCTTTCCTCACCGGGGCTTGCTGTTGGATACATCTCGCCATTACCTGCCACTCTCTAGCATCCTGGACACTCTGGATGTCATGGCGTACAATAAATTGAACGTGTTCCACTGGCATCTGGTAGATGATCCTTCCTTCCCATATGAGAGCTTCACTTTTCCAGAGCTCATGAGAAAGGGGTCCTACAACCCTGTCACCCACATCTACACAGCACAGGATGTGAAGGAGGTCATTGAATACGCACGGCTCCGGGGTATCCGTGTGCTTGCAGAGTTTGACACTCCTGGCCACACTTTGTCCTGGGGACCAGGTATCCCTGGATTACTGACTCCTTGCTACCCTGGGTCTGAGCCCTCTGGCACCTTTGGACCAGTGAATCCCAGTCTCAATAATACCTATGAGTTCATGAGCACATTCTTCTTAGAAGTCAGCTCTGTCTTCCCAGATTTTTATCTTCATCTTGGAGGAGATGAGGTTGATTTCACCTGCTGGAAGTCCAACCCAGAGATCCAGGACTTTATGAGGAAGAAAGGCTTCGGTGAGGACTTCAAGCAGCTGGAGTCCTTCTACATCCAGACGCTGCTGGACATCGTCTCTTCTTATGGCAAGGGCTATGTGGTGTGGCAGGAGGTGTTTGATAATAAAGTAAAGATTCAGCCAGACACAATCATACAGGTGTGGCGAGAGGATATTCCAGTGAACTATATGAAGGAGCTGGAACTGGTCACCAAGGCCGGCTTCCGGGCCCTTCTCTCTGCCCCCTGGTACCTGAACCGTATATCCTATGGCCCTGACTGGAAGGATTTCTACGTAGTGGAACCCCTGGCATTTGAAGGTACCCCTGAGCAGAAGGCTCTGGTGATTGGTGGAGAGGCTTGTATGTGGGGAGAATATGTGGACAACACAAACCTGGTCCCCAGGCTCTGGCCCAGAGCAGGGGCTGTTGCCGAAAGGCTGTGGAGCAACAAGTTGACATCTGACCTGACATTTGCCTATGAACGTTTGTCACACTTCCGCTGTGAGTTGCTGAGGCGAGGTGTCCAGGCCCAACCCCTCAATGTAGGCTTCTGTGAGCAGGAGTTTGAACAGACCTGCCCAACTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTTGCAACGAAC.

The corresponding HEXA protein has the following amino acid sequence:

(SEQ ID NO: 370) MTSSRLWFSLLLAAAFAGRATALWPWPQNFQTSDQRYVLYPNNFQFQYDVSSAAQPGCSVLDEAFQRYRDLLFGSGSWPRPYLTGKRHTLEKNVLVVSVVTPGCNQLPTLESVENYTLTINDDQCLLLSETVWGALRGLETFSQLVWKSAEGTFFINKTEIEDFPRFPHRGLLLDTSRHYLPLSSILDTLDVMAYNKLNVFHWHLVDDPSFPYESFTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRGIRVLAEFDTPGHTLSWGPGIPGLLTPCYPGSEPSGTFGPVNPSLNNTYEFMSTFFLEVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQLESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDIPVNYMKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYVVEPLAFEGTPEQKALVIGGEACMWGEYVDNTNLVPRLWPRAGAVAERLWSNKLTSDLTFAYERLSHFRCELLRR GVQAQPLNVGFCEQEFEQT.

Notably, the resulting RNA motifs would be included within thetranslated region of the HEXA gene, disrupting the function of theprotein coding gene. Inserted polyadenylation motifs would result inpremature transcript termination. This site is merely illustrative ofthe potential PEgRNAs that could result in insertion of the listed RNAmotifs of the above table within a genomic site that is transcribed andthus which would produce an RNA product.

PEgRNAs for use with PE for RNA tagging could be expressed from a U6promoter (in which case a single guanosine would be added to the 5′ endof the PEgRNA for guides that include protospacers that do not beginwith a G and 6-7 thymine would be added to the 3′ end) or a pol IIpromoter such as pCMV (in which case it might be necessary to remove theintrinsically transcribed sequence of this promoter from the 5′ end ofthe RNA via a self-cleaving element or Csy4 motif, and a terminationmotif would need to be added to the 3′ end of the RNA that does notresult in export of the RNA from the nucleus, such as the 3′ box motiflisted above. Note that this motif would not be inserted into the genomeas a result of PE, as it would be 3′ of the annealing region). The corePEgRNA scaffold is underlined, the homology and annealing regions areitalicized, and the inserted sequence is bolded. Note that the sequenceinserted is the reverse complement of the above examples—as describedbelow and therefore these PEgRNAs would need to be targeted to thecoding strand.

Also, note that self-cleaving ribozymes other than HDV need in someembodiments to be tailored to the given target site; that is, while HDVcleaves the encoded transcript immediately 5′ to itself, the cut sitesfor all other self-cleaving ribozymes are within the ribozyme itself.Therefore, the first and last roughly 5-10 nucleotides (and in someinstances potentially more than 10) would actually be a part of theencoded sequence. As an example, to cleave the sequence5′-NNNNNTCATCCTGATAAACTGCAAA-3′ (SEQ ID NO: 371) after the 5 Ns, where Nis any nucleotide, using a hammerhead self-cleaving ribozyme, thefollowing sequence would be inserted, where the underlined sequencesform an imperfect RNA pairing element.

(SEQ ID NO: 372) 5′NNNNNCAGTTTGTACGGATGACTGATGAGTCCCAAATAGGACGAAACGCGCTTCGGTGCGTCTCATCCTGATAAACTGCAAA-3′.

There is significant flexibility in terms of the length and nature ofthis pairing element, and this would be true for any of the non-HDVself-cleaving ribozymes listed in the original submission. To install ahammerhead ribozyme to cleave the hexA mRNA using a PEgRNA with the sameprotospacer as the above listed constructs, the following PEgRNAsequence could be used (labels same as above):

(SEQ ID NO: 373) 5′ACCTGAACCGTATATCGACGCACCGAAGCGCGTTTCGTCCTATTTGGGACTCATCAGGATATACGGTTCAGGTGATATACGGTTCAGGTGACGCACCGAAGCGCGTTTCGTCCTATTTGGGACTCATCAGACCTGAACCGTATATCATCCTTCCAGTCAGGGCCATGTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCACCTGAACCGTATATCGACGCACCGAAGCGCGTTTCGTCCTATTTGGGACTCATCAGGATATACGGTTCAGGTCTATGGCCCTGACTGGAA-3′(wherein the core PEgRNA scaffold is underlined,the homology and annealing regions are italicized,and the inserted sequence is bolded).

Designing other PEgRNA for insertion of RNA motifs may follow thegeneral principle described herein. However, it is noted that many ofRNA motifs are potentially highly structured, which could make itdifficult for them to be reverse-transcribed and inserted into thegenome. Although for some RNA sequences, such as simple hairpins, boththe RNA sequence itself and its complement are structured. However, thatis unlikely to be true for the sequences noted above. Therefore, wheninserting these motifs, it would most likely be best for the PEgRNA toencode the reverse complement of these sequences, resulting in theinsertion of the DNA sequence actually encoding the motif into thegenome. Similarly, inclusion of a self-cleaving ribozyme in the PEgRNAtemplate region would result in processing and inefficient activity,while inclusion of its reverse complement would not. Thus, these PEgRNAswill likely have to target the coding strand, whereas PEgRNAs encodingother types of insertions (such as therapeutic correction) would be ableto theoretically target either strand.

Also, note that for many of the inserted motifs, the resulting PEgRNAmight not be able to be transcribed from the U6 promoter, necessitatinguse of other promoters, such as pCMV. Similarly, longer PEgRNAs couldalso be less stable. Shorter motifs, such as m⁶A markers, would not havethis challenge.

G. Use of Prime Editing for Generation of Sophisticated Gene Libraries

Prime editing may also be used to generate sophisticated libraries ofprotein- or RNA-coding genes with defined or variable insertions,deletions, or defined amino acid/nucleotide conversions, and their usein high-throughput screening and directed evolution is described herein.This application of prime editing can be further described in Example 7.

The generation of variable genetic libraries has most commonly beenaccomplished through mutagenic PCR (see Cadwell R C and Joyce G F. PCRMethods Appl. 1992). This method relies on either using reactionconditions that reduce the fidelity of DNA polymerase, or using modifiedDNA polymerases with higher mutation rates. As such, biases in thesepolymerases are reflected in the library product (e.g. a preference fortransition mutations versus transversions). An inherent limitation ofthis approach to library construction is a relative inability to affectthe size of the gene being varied. Most DNA polymerases have extremelylow rates of indel mutations (insertions or deletions), and most ofthese will result in frameshift mutations in protein-coding regions,rendering members of the library unlikely to pass any downstreamselection (See McInerney P, Adams P, and Hadi M Z. Mol Biol Int. 2014).

Additionally, biases in PCR and cloning can make it difficult togenerate single libraries consisting of genes of different sizes. Theselimitations can severely limit the efficacy of directed evolution toenhance existing or engineer novel protein functions. In naturalevolution, large changes in protein function or efficacy are typicallyassociated with insertion and deletion mutations that are unlikely tooccur during canonical library generation for mutagenesis. Furthermore,these mutations most commonly occur in regions of the protein inquestion that are predicted to form loops, as opposed to the hydrophobiccore. Thus, most indels generated using a traditional unbiased approachare likely to either be deleterious or ineffective.

Libraries that could bias such mutations to the sites within the proteinwhere they would be most likely to be beneficial, e.g., loop regions,would have a significant advantage over traditional libraries given thatall libraries access only a fraction of the possible mutation space.Finally, although it is possible to generate genetic libraries withsite-specific indel mutations through multistep PCR and clonal assemblyusing NNK primers or via DNA shuffling, these libraries cannot undergoadditional rounds of ‘indelgenesis’ in continual evolution. Continuousevolution is a type of directed evolution with minimal userintervention. One such example is PACE (see Esvelt K M, Carlson J C, andLiu D R. Nature. 2011). Because continuous evolution occurs with minimaluser intervention, any increase in library diversity during theevolution must occur using the native replication machinery. As such,although libraries of genes with inserted or removed codons as specificloci can be generated and screened in PACE, additional rounds of‘indelgenesis’ are not possible.

It is envisioned that the programmability of prime editing (PE) can beleveraged to generate highly sophisticated, programmed genetic librariesfor use in high-throughput screening and directed evolution (see FIG.29A). PE can insert, change or remove defined numbers of nucleotidesfrom specified genetic loci using information encoded in a prime editingguide RNA (PEgRNA) (see FIG. 29B). This enables the generation oftargeted libraries with one or more amino acids inserted or removed fromthe loop regions wherein mutations are most likely to give rise tochanges in function, without background introduction of nonfunctionalframeshift mutations (see FIG. 29C). PE can be used to install specificsets of mutations without regard for biases inherent in either DNApolymerase or the sequence being mutated.

For instance, while converting a CCC codon to a stop codon would be anunlikely occurrence via canonical library generation because it wouldrequire three consecutive mutations, including two transversions, PEcould be used to convert any given, targeted codon to a TGA stop codonin one step. They could also be used to install programmed diversity atgiven positions, for instance by incorporating codons encoding anyhydrophobic amino acid at a given site, while not encoding any others.Furthermore, because of the programmability of PE, multiple PEgRNAscould be utilized to generate multiple different edits at multiple sitessimultaneously, enabling the generation of highly programmed libraries(see FIG. 29D). Additionally, it is possible to use reversetranscriptases with lower fidelity to generate regions of mutagenesiswithin an otherwise invariable library (such as the HIV-I reversetranscriptase or Bordetella phage reverse transcriptase) (see Naorem SS, Hin J, Wang S, Lee W R, Heng X, Miller J F, Guo H. Proc Natl Acad Sci2017 and Martinez M A, Vartanian J P, Wain-Hobson S. Proc Natl Acad SciUSA 1994).

The possibility of iterative rounds of PE on the same site is alsoenvisioned, allowing—for instance—the repeated insertion of codons at asingle site, e.g., in a loop region. Also, it is envisioned that all ofthe above described approaches can be incorporated into continualevolution, enabling the generation of novel in situ evolving libraries(see FIG. 30 ). They could also be used to construct these librarieswithin other cell types where it would otherwise be difficult toassemble large libraries, for instance within mammalian cells.Generation of PE-encoding bacterial strains that have been optimized fordirected evolution would be a useful additional tool for theidentification of proteins and RNAs with improved or novelfunctionality. All of these uses of PE are non-obvious due to the novelnature of PEs. In conclusion, library generation via PE would be ahighly useful tool in synthetic biology and directed evolution, as wellas for high-throughput screening of protein and RNA combinatorialmutants.

Competing Approaches

The chief method by which diverse libraries are currently generated isby mutagenic PCR (see Cadwell R C and Joyce G F. PCR Methods Appl.1992), described above. Insertions or deletions can be introduced viadegenerated NNK primers at defined sites during PCR, althoughintroducing such mutations at multiple sites requires multiple rounds ofiterative PCR and cloning before constructing a more diverse library viamutagenic PCR, rendering the method slow. An alternative, complementarymethod is DNA shuffling, where fragments of a library of genes generatedvia DNase treatment are introduced into a PCR reaction without primers,resulting in the annealing of different fragments to each other and therapid generation of more diverse libraries than via mutagenic PCR alone(see Meyer A J, Ellefson J W, Ellington A D. Curr Protoc Mol Biol.2014). Although this approach can theoretically generate indelmutations, it more often results in frameshift mutations that destroygene function. Furthermore, DNA shuffling requires a high degree ofhomology between gene fragments.

Both of these methods must be done in vitro, with the resulting librarytransformed into cells, while libraries generated by PE can beconstructed in situ, enabling their use in continual evolution. Whilelibraries can be constructed in situ through in vivo mutagenesis, theselibraries rely on the host cellular machinery and exhibit biases againstindels. Similarly, although traditional cloning methods can be used togenerate site-specific mutational profiles, they cannot be used in situand are generally assembled one at a time in vitro before beingtransformed into cells. The efficiency and broad functionality of PE inboth prokaryotic and eukaryotic cell types further suggests that theselibraries could be constructed directly in the cell type of interest, asopposed to being cloned into a model organism such as E. coli and thentransferred into the cell or organism of interest. Another competingapproach for targeted diversification is automated multiplex genomeengineering, or MAGE, wherein multiple single-stranded DNAoligonucleotides can be incorporated within replication forks and resultin programmable mutations⁷. However, MAGE requires significantmodification of the host strain and can lead to a 100-fold increase inoff-target or background mutations (see Nyerges Á et al. Proc Natl AcadSci USA. 2016), whereas PE is more highly programmed and anticipated toresult in fewer off-target effects. Additionally, MAGE has not beendemonstrated in a wide variety of cell types, including mammalian cells.

By contrast, prime editing is a novel and non-obvious complementarytechnique for library generation.

Use of PE for Constructing Gene Libraries

PE may be used to construct gene libraries in a programmable manner.

In one example, PE can be used in a directed evolution experiment tointroduce protein variants into gene libraries during a continualevolution experiment using PACE, permitting iterative accumulation ofboth point mutations and indels in a manner not possible via traditionalapproaches.

It has already been shown that PE can site-specifically and programmablyinsert nucleotides into a genetic sequence in E. coli. Directedevolution can be used to identify monobodies with improved binding to aspecific epitope via a modified two-hybrid protein:protein binding PACEselection. Specific and highly variable loops within these monobodiescontribute significantly to affinity and specificity. Improved monobodybinding might be obtained rapidly in PACE by varying the length andcomposition of these loops in a targeted fashion. However, varyingsequence length is not an established functionality of PACE. Whilelibrary of varied loop sizes might be used as a starting point for PACE,no subsequent improvements to length would arise throughout the PACEselection, barring access to beneficial synergistic combinations ofpoint mutations and indel mutations.

In various embodiments, PE can be used to improve the PACE selection byenabling the in situ generation and evolution of monobodies with varyingloop lengths. To do so, the PACE E. coli strain may be introduced to anadditional PE plasmid, which encodes the PE enzyme and one or morePEgRNAs. Expression of PE enzyme and PEgRNAs in the E. coli would beunder the control of a small molecule delivered to the PACE lagoon at arate selected by the experimenter.

In various embodiments, the PEgRNA components would contain a spacerdirecting the PE to the site of interest on the selection phage andwould be designed such that a multiple of three nucleotides could beinserted at the target site such that a new PEgRNA binding site would beintroduced, enabling the iterative insertion of one or more codons atthe targeted site.

In parallel, another host E. coli strain might include PEgRNAs thatwould template the removal of one or more codons, enabling loop size toshrink during the evolution. A PACE experiment might utilize a mixtureof both strains or alternate the two to permit the slow and controlledaddition or removal of loop sequences.

In addition to the use of PE and PACE to create monobody libraries, thistechnique can also be applied to the evolution of antibodies using PEand PACE. The binding principles governing antibodies are very similarto those governing monobodies: the length of antibodycomplement-determining region loops is critical to their bindingfunction. Further, longer loop lengths have been found to be critical inthe development of rare antibodies with broadly protective activityagainst HIV-1 and other viral infections (see Mascola J R, Haynes B F.Immunol Rev. 2013). Application of PE as described above to an antibodyor antibody-derived molecule would permit the generation of antibodieswith diverse loop length and varied loop sequence. In combination withPACE, such an approach would permit enhanced binding through loopgeometries not accessible to standard PACE, and thus permit evolution ofhighly functional antibodies.

As a non-limiting example, the following PEgRNAs could be used toprogrammably modify the genome of a bacteriophage used in a continuousevolution experiment:

MODIFICATION PEGRNA SEQUENCE CCA UACACCAUCACGGUCUAUGCGUUUUAGAGCUAGAAAINSERTION UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUAUCUUCGCCCC AUGCAUAGACCGUGAUGG (SEQ ID NO: 101)1 NT DELETION CGCGUCGCGCUCGUCAGAUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUCCGCCUACC UGCAUCUGACGAGCGCGA (SEQ ID NO: 102)POINT AUCGGAGAAUACAUGAACAUGUUUUAGAGCUAGAAA MUTATIONUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCCGGAGAAUACAUGAACAUCGGACCCGCGCUAUCUUC (SEQ ID NO: 103) NNNUACACCAUCACGGUCUAUGCGUUUUAGAGCUAGAAA INSERTIONUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU (N = A/T/G/C)GAAAAAGUGGCACCGAGUCGGUGCUAUCUUCGCCNN NUGCAUAGACCGUGAUGG (SEQ ID NO: 104)ITERATIVE UACACCAUCACGGUCUAUGCGUUUUAGAGCUAGAAA GGGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU INSERTIONGAAAAAGUGGCACCGAGUCGGUGCGGGGGGGGGGGG GUGCAUAGACCGUGAUGG (SEQ ID NO: 105)

In various embodiments, the use of PE for constructing gene librariesmay make the use of the mutagenic activity of error-prone reversetranscriptases. The use of such mutagenic reverse transcriptase mayfacilitate the generation of mutagenized programmable libraries due tothe lower fidelity of the error-prone RTs. As used herein, the term“error-prone” reverse transcriptase refers to a reverse transcriptaseenzyme that occurs naturally or which has been derived from anotherreverse transcriptase (e.g., a wild type M-MLV reverse transcriptase)which has an error rate that is less than the error rate of wild typeM-MLV reverse transcriptase. The error rate of wild type M-MLV reversetranscriptase is reported to be in the range of one error in 15,000 to27,000 nucleobase incorporations. See Boutabout et al. (2001) “DNAsynthesis fidelity by the reverse transcriptase of the yeastretrotransposon Ty1,” Nucleic Acids Res 29(11):2217-2222, which isincorporated herein by reference.

Thus, for purposes of this application, the term “error prone” refers tothose RT that have an error rate that is greater than one error in15,000 nucleobase incorporation (6.7×10⁻⁵ or higher), e.g., 1 error in14,000 nucleobases (7.14×10⁻⁵ or higher), 1 error in 13,000 nucleobasesor fewer (7.7×10⁻⁵ or higher), 1 error in 12,000 nucleobases or fewer(7.7×10⁻⁵ or higher), 1 error in 11,000 nucleobases or fewer (9.1×10⁻⁵or higher), 1 error in 10,000 nucleobases or fewer (1×10⁻⁴ or 0.0001 orhigher), 1 error in 9,000 nucleobases or fewer (0.00011 or higher), 1error in 8,000 nucleobases or fewer (0.00013 or higher) 1 error in 7,000nucleobases or fewer (0.00014 or higher), 1 error in 6,000 nucleobasesor fewer (0.00016 or higher), 1 error in 5,000 nucleobases or fewer(0.0002 or higher), 1 error in 4,000 nucleobases or fewer (0.00025 orhigher), 1 error in 3,000 nucleobases or fewer (0.00033 or higher), 1error in 2,000 nucleobase or fewer (0.00050 or higher), or 1 error in1,000 nucleobases or fewer (0.001 or higher), or 1 error in 500nucleobases or fewer (0.002 or higher), or 1 error in 250 nucleobases orfewer (0.004 or higher).

A variety of mutagenic RTs could be envisioned for generation of highlymutagenized programmable libraries. Two such examples are the mutagenicreverse transcriptases from Bordetella phage (see Handa, S., et al. NuclAcids Res 9711-25 (2018), which is incorporated herein by reference) andLegionella pneumophila (see Arambula, D., et al. Proc Natl Acad Sci USA8212-7 (2013), which is incorporated by reference). In the case of theRT from Bordetella phage (brt), an accessory protein might need to alsobe added (bavd) to Cas9—or delivered in trans—as well as additional RNAsequences to the PEgRNA to improve binding of the mutagenic RT to thetarget site (see Handa, S., et al. Nucl Acids Res 9711-25 (2018)). Whenusing mutagenic RTs, the template region of the PEgRNA might be enrichedin adenosines or AAY codons to enhance diversity.

The amino acid sequence of the mutagenic RT from Bordetella phage isprovided as follows. Like other RTs disclosed herein, the Brt proteinmay be fused to a napDNAbp as a fusion protein to form a functional PE.

Name Sequence brt MGKRHRNLIDQITTWENLLDAYRKTSHGKRRTWGYLEF mutagenicKEYDLANLLALQAELKAGNYERGPYREFLVYEPKPRLI rtSALEFKDRLVQHALCNIVAPIFEAGLLPYTYACRPDKGTHAGVCHVQAELRRTRATHFLKSDFSKFFPSIDRAALYAMIDKKIHCAATRRLLRVVLPDEGVGIPIGSLTSQLFANVYGGAVDRLLHDELKQRHWARYMDDIVVLGDDPEELRAVFYRLRDFASERLGLKISHWQVAPVSRGINFLGYRIWPTHKLLRKSSVKRAKRKVANFIKHGEDESLQRFLASWSGHAQWADTHNLFTWMEEQYGIACH (SEQ ID NO: 129)

In the case of Brt from Bordetella, the PE fusion may also include anadditional accessory protein (Bavd). The accessory protein may be fusedto the PE fusion protein or provided in trans. The amino acid sequenceof Bavd accessory protein is provided as follows:

Name Sequence bavd MEPIEEATKCYDQMLIVERYERVISYLYPIAQSIPRKHGV accessoryAREMFLKCLLGQVELFIVAGKSNQVSKLYAADAGLAMLRF proteinWLRFLAGIQKPHAMTPHQVETAQVLIAEVGRILGS to brtWIARVNRKGQAGK (SEQ ID NO: 236)

In the case of Brt from Bordetella, the PEgRNA may comprise anadditional nucleotide sequence added a PEgRNA, e.g., to the 5′ or 3′end. Exemplary sequence is as follows, which is originally from theBordetella phage genome:

NAME SEQUENCE PEGRNA- ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGC ADDITIONUGGCCUUUCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCU 1UCGGCGAACACGUUACACGGUUCGGCAAAACGUCGAUUACUGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCUGGGA AAUAA (SEQ ID NO: 237)

This PEgRNA addition sequence can be reduced in various ways to shortenthe length. For example, the PEgRNA-addition 1 sequence could be reducedto the following exemplary alternative addition sequences:

NAME SEQUENCE PEGRNA- ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGG ADDITIONCUGGCCUUUCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUG 2CUUCGGCGAACACGUUACACGGUUCGGCAAAACGUCGAUUACUGAAAAUGGAAAGGCGGGGCCGACUUC (SEQ ID NO: 238) PEGRNA-ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGG ADDITIONCUGGCCUUUCCUCAAUAGGUGGUCAAAGGGCAGGCUGGGA 3 AAUAA (SEQ ID NO: 239)PEGRNA- ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGG ADDITIONCUGGCCUUUCCUCAAUAGGUGGUCA (SEQ ID NO: 4 277) PEGRNA-CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUU ADDITIONCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGCGA 5ACACGUUACACGGUUCGGCAAAACGUCGAUUACUGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCUGGGAAAUAA (SEQ ID NO: 240) PEGRNA-CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUU ADDITIONCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGCGA 6ACACGUUACACGGUUCGGCAAAACGUCGAUUACUGAAAAUGGAAAGGCGGGGCCGACUUC (SEQ ID NO: 241) PEGRNA-CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUU ADDITIONCCUCAAUAGGUGGUCAAAGGGCAGGCUGGGAAAUAA 7 (SEQ ID NO: 242) PEGRNA-CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUU ADDITION CCUCAAUAGGUGGUCA 8(SEQ ID NO: 243)

In other embodiments, the PEgRNA addition sequence can be also bemutated. For example, the PEgRNA-addition 1 sequence could be mutated tothe following exemplary alternative addition sequence:

NAME SEQUENCE PEGRNA- ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGC ADDITIONUGGCCUUUCCUCAAUAGAUGAGCCGCCGGUUCUGUCCUGCU 1UCGGCGAACACGUUACACGGUUCGGCAAAACGUCGAUUACU MUTATEDGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCUGGGA AAUAA (SEQ ID NO: 244)

In various embodiments relating to the use of PE for designing genelibraries, special PEgRNAs considerations may apply. For example,without wishing to be bound by theory, the additional PEgRNA sequencesdescribed above might be needed to enable efficient mutagenesis viamutagenic RTs. In another embodiment, iterative codon insertion using PEmay required specific PEgRNA designs. For example, to insert a GGG(glycine) codon iteratively, the entire homology region of the PEgRNAmight need to be composed of Gs, as shown above. This would mean thatonly certain sites could go iterative insertion. Additionally, the PAMsequence would not be able to be disrupted by the PE.

H. Use of Prime Editing for Insertion of Immunoepitopes

Prime editors may also be used as a means to insert known immunogenicityepitopes into endogenous or foreign genomic DNA, resulting inmodification of the corresponding proteins for therapeutic orbiotechnological applications (see FIGS. 31 and 32 ). Prior to theinvention of prime editing, such insertions could be achieved onlyinefficiently and with high rates of indel formation from DSBs. primeediting solves the problem of high indel formation from insertion editswhile generally offering higher efficiency than HDR. This lower rate ofindel formation presents a major advantage of prime editing over HDR asa method for targeted DNA insertions, especially in the describedapplication of inserting immunogenicity epitopes. The length of epitopesis in a range from few bases to hundreds of bases. Prime editor is anefficient approach to achieve such targeted insertions in mammaliancells.

The key concept of the invention is the use of prime editors to insert anucleotide sequence containing previously described immunogenicityepitopes into endogenous or foreign genomic DNA for the downregulationand/or destruction of their protein products and/or expressing celltypes. Nucleotide sequences for immunogenic epitope insertion would betargeted to genes in a manner to produce fusion proteins of the targetedgene's coded protein and the inserted immunogenic epitope'scorresponding protein translation. Patient's immune systems will havebeen previously trained to recognize these epitopes as a result ofstandard prior immunization from routine vaccination against, forexample, tetanus or diphtheria or measles. As a result of theimmunogenic nature of the fused epitopes, patient's immune systems wouldbe expected to recognize and disable the prime edited protein (not justthe inserted epitope) and potentially the cells from which it wasexpressed.

Precise genome targeting technologies using the CRISPR/Cas system haverecently been explored in a wide range of applications, including theinsertion of engineered DNA sequences into targeted genomic loci.Previously, homology-directed repair (HDR) has been used for thisapplication, requiring an ssDNA donor template and repair initiation bymeans of a double-stranded DNA break (DSB). This strategy offers thebroadest range of possible changes to be made in cells and is the onlymethod available to insert large DNA sequences into mammalian cells.However, HDR is hampered by undesired cellular side effects stemmingfrom its initiating DSB, such as high levels of indel formation, DNAtranslocations, large deletions, and P53 activation. In addition tothese drawbacks, HDR is limited by low efficiency in many cell types (Tcells are a notable exception to this observation). Recent efforts toovercome these drawbacks include fusing human Rad51 mutants to a Cas9D10A nickase (RDN), resulting in a DSB-free HDR system that featuresimproved HDR product:indel ratios and lower off target editing, but isstill hampered by cell-type dependencies and only modest HDR editingefficiency.

Recently developed fusions of Cas9 to reverse transcriptases (“Primeeditors”) coupled with PEgRNAs represent a novel genome editingtechnology that offers a number of advantages over existing genomeediting methods, including the ability to install any single nucleotidesubstitution, and to insert or delete any short stretch of nucleotides(up to at least several dozen bases) in a site-specific manner. Notably,PE edits are achieved with generally low rates of unintended indels. Assuch, PE enables targeted insertion-based editing applications that havebeen previously impossible or impractical.

This particular aspect describes a method for using prime editing as ameans to insert known immunogenicity epitopes into endogenous or foreigngenomic DNA, resulting in modification of the corresponding proteins fortherapeutic or biotechnological applications (see FIGS. 31 and 32 ).Prior to the invention of prime editing, such insertions could beachieved only inefficiently and with high rates of indel formation fromDSBs. prime editing solves the problem of high indel formation frominsertion edits while generally offering higher efficiency than HDR.This lower rate of indel formation presents a major advantage of primeediting over HDR as a method for targeted DNA insertions, especially inthe described application of inserting immunogenicity epitopes. Thelength of epitopes is in a range from few bases to hundreds of bases.Prime editor is the most efficient and cleanest technology to achievesuch targeted insertions in mammalian cells.

The key concept of this aspect is the use of prime editors to insert anucleotide sequence containing previously described immunogenicityepitopes into endogenous or foreign genomic DNA for the downregulationand/or destruction of their protein products and/or expressing celltypes. Nucleotide sequences for immunogenic epitope insertion would betargeted to genes in a manner to produce fusion proteins of the targetedgene's coded protein and the inserted immunogenic epitope'scorresponding protein translation. Patient's immune systems will havebeen previously trained to recognize these epitopes as a result ofstandard prior immunization from routine vaccination against, forexample, tetanus or diphtheria or measles. As a result of theimmunogenic nature of the fused epitopes, patient's immune systems wouldbe expected to recognize and disable the prime edited protein (not justthe inserted epitope) and potentially the cells from which it wasexpressed.

Fusions to targeted genes would be engineered as needed to ensure theinserted epitope protein translation is exposed for immune systemrecognition. This could include targeted nucleotide insertions resultingin protein translations yielding C-terminal fusions of immunogenicityepitopes to targeted genes, N-terminal fusions of immunogenicityepitopes to targeted genes, or the insertion of nucleotides into genesso that immunogenicity epitopes are coded within surfaced-exposedregions of protein structure.

Protein linkers encoded as nucleotides inserted between the target genesequence and the inserted immunogenicity epitope nucleotide sequence mayneed to be engineered as part of this invention to facilitate immunesystem recognition, cellular trafficking, protein function, or proteinfolding of the targeted gene. These inserted nucleotide-encoded proteinlinkers may include (but are not limited to) variable lengths andsequences of the XTEN linker or variable lengths and sequences ofGlycine-Serine linkers. These engineered linkers have been previous usedto successfully facilitate protein fusions. Exemplary linkers mayinclude any of those described herein, including the amino acid sequence(GGGGS)n (SEQ ID NO: 165), (G)n (SEQ ID NO: 166), (EAAAK)n (SEQ ID NO:167), (GGS)n (SEQ ID NO: 168), (SGGS)n (SEQ ID NO: 169), (XP)n (SEQ IDNO: 170), or any combination thereof, wherein n is independently aninteger between 1 and 30, and wherein X is any amino acid. In someembodiments, the linker comprises the amino acid sequence (GGS)n (SEQ IDNO: 176), wherein n is 1, 3, or 7. In some embodiments, the linkercomprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 171). Insome embodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 172). In some embodiments,the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO:173). In some embodiments, the linker comprises the amino acid sequenceSGGS (SEQ ID NO: 174).

Distinguishing features of this aspect include the ability to usepreviously acquired immune responses to specific amino acid sequences asa means to induce an immune response against otherwise non-immunogenicproteins. Another distinguishing feature is the ability to insert thenucleotide sequences of these immunogenic epitopes in a targets mannerthat does not induce high levels of unwanted indels as a by-productediting and is efficient in its insertion. This specific application ofPE has the ability to combine cell type-specific delivery methods (suchas AAV serotypes) to insert epitopes in cell types that are of interestto trigger an immune response to.

Prime editing as a means of inserting immunogenic epitopes intopathogenic genes could be used to program the patient's immune system tofight a wide variety of diseases (not limited to cancer as withimmuno-oncology strategies). An immediately relevant use of thistechnology would be as a cancer therapeutic as it could undermine atumor's immune escape mechanism by causing an immune response to arelevant oncogene like HER2 or growth factors like EGFR. Such anapproach could seem similar to T-cell engineering, but one novel advanceof this approach is that it can be utilized in many cell types and fordiseases beyond cancer, without needed to generate and introduceengineered T-cells into patients.

Using PE to insert an immunogenicity epitope which most people arealready vaccinated against (tetanus, pertussis, diphtheria, measles,mumps, rubella, etc.) into a foreign or endogenous gene that drives adisease, so the patient's immune system learns to disable that protein.

Diseases that stand to have a potential therapeutic benefit from theaforementioned strategy include those caused by aggregation of toxicproteins, such as in fatal familial insomnia. Other diseases that couldbenefit include those caused by pathogenic overexpression of anotherwise nontoxic endogenous protein, and those caused by foreignpathogens.

Primary therapeutic indications include those mentioned above such astherapeutics for cancer, prion and other neurodegenerative diseases,infectious diseases, and preventative medicine. Secondary therapeuticindications may include preventative care for patients with late-onsetgenetic diseases. It is expected that current standard of care medicinesmay be used in conjunction with prime editing for some diseases, likeparticularly aggressive cancers, or in cases where medications helpalleviate disease symptoms until the disease completely cured. Below areexamples of immunoepitopes that may be inserted into genes by the hereindisclosed prime editors:

EPITOPE AMINO EXAMPLE NUCLEIC ACID VACCINE DISEASE ACID SEQUENCESEQUENCE (8) 1 TETANUS QYIKANSKFIGITE CATGATATAAAAGCAAATTCTAAATTTATOXOID L (SEQ ID NO: 396) TAGGTATAACTGAACTA (SEQ ID NO: 397) 2DIPHTHERIA GADDVVDSSKSF GGCGCCGACGACGTGGTGGACAGCAG TOXIN VMENFSSYHGTKCAAGAGCTTCGTGATGGAGAACTTCA MUTANT PGYVDSIQKGIQKGCAGCTACCACGGCACCAAGCCCGGC CRM197 PKSGTQGNYDDDTACGTGGACAGCATCCAGAAGGGCAT WKEFYSTDNKYD CCAGAAGCCCAAGAGCGGCACCCAGGAAGYSVDNENPL GCAACTACGACGACGACTGGAAGGAG SGKAGGVVKVTYTTCTACAGCACCGACAACAAGTACGA PGLTKVLALKVD CGCCGCCGGCTACAGCGTGGACAACGNAETIKKELGLSL AGAACCCCCTGAGCGGCAAGGCCGGC TEPLMEQVGTEEFGGCGTGGTGAAGGTGACCTACCCCGG IKRFGDGASRVVL CCTGACCAAGGTGCTGGCCCTGAAGGSLPFAEGSSSVEYI TGGACAACGCCGAGACCATCAAGAAG NNWEQAKALSVEGAGCTGGGCCTGAGCCTGACCGAGCC LEINFETRGKRGQ CCTGATGGAGCAGGTGGGCACCGAGGDAMYEYMAQAC AGTTCATCAAGAGGTTCGGCGACGGC AGNRVRRSVGSSLGCCAGCAGGGTGGTGCTGAGCCTGCC SCINLDWDVIRDK CTTCGCCGAGGGCAGCAGCAGCGTGGTKTKIESLKEHGPI AGTACATCAACAACTGGGAGCAGGCC KNKMSESPNKTVAAGGCCCTGAGCGTGGAGCTGGAGAT SEEKAKQYLEEFH CAACTTCGAGACCAGGGGCAAGAGGGQTALEHPELSELK GCCAGGACGCCATGTACGAGTACATGG TVTGTNPVFAGACCCAGGCCTGCGCCGGCAACAGGGTG NYAAWAVNVAQV AGGAGGAGCGTGGGCAGCAGCCTGAGIDSETADNLEKTT CTGCATCAACCTGGACTGGGACGTGAT AALSILPGIGSVMCAGGGACAAGACCAAGACCAAGATCG GIADGAVHHNTEE AGAGCCTGAAGGAGCACGGCCCCATCIVAQSIALSSLMVA AAGAACAAGATGAGCGAGAGCCCCAA QAIPLVGELVDIGFCAAGACCGTGAGCGAGGAGAAGGCC AAYNFVESIINLFQ AAGCAGTACCTGGAGGAGTTCCACCAVVHNSYNRPAYSP GACCGCCCTGGAGCACCCCGAGCTGA GHKTQPFLHDGYGCGAGCTGAAGACCGTGACCGGCACC AVSWNTVEDSIIR AACCCCGTGTTCGCCGGCGCCAACTATGFQGESGHDIKI CGCCGCCTGGGCCGTGAACGTGGCCC TAENTPLPIAGVLAGGTGATCGACAGCGAGACCGCCGAC LPTIPGKLDVNKS AACCTGGAGAAGACCACCGCCGCCCTKTHISVNGRKIRM GAGCATCCTGCCCGGCATCGGCAGCGT RCRAIDGDVTFCRGATGGGCATCGCCGACGGCGCCGTGC PKSPVYVGNGVH ACCACAACACCGAGGAGATCGTGGCCANLHVAFHRSSSE CAGAGCATCGCCCTGAGCAGCCTGAT KIHSNEISSDSIGVGGTGGCCCAGGCCATCCCCCTGGTGG LGYQKTVDHTKV GCGAGCTGGTGGACATCGGCTTCGCCNSKLSLFFEIKS GCCTACAACTTCGTGGAGAGCATCATC (SEQ ID NO: 398)AACCTGTTCCAGGTGGTGCACAACAG CTACAACAGGCCCGCCTACAGCCCCGGCCACAAGACCCAGCCCTTCCTGCAC GACGGCTACGCCGTGAGCTGGAACACCGTGGAGGACAGCATCATCAGGACCG GCTTCCAGGGCGAGAGCGGCCACGACATCAAGATCACCGCCGAGAACACCCC CCTGCCCATCGCCGGCGTGCTGCTGCCCACCATCCCCGGCAAGCTGGACGTGA ACAAGAGCAAGACCCACATCAGCGTGAACGGCAGGAAGATCAGGATGAGGTG CAGGGCCATCGACGGCGACGTGACCTTCTGCAGGCCCAAGAGCCCCGTGTAC GTGGGCAACGGCGTGCACGCCAACCTGCACGTGGCCTTCCACAGGAGCAGCA GCGAGAAGATCCACAGCAACGAGATCAGCAGCGACAGCATCGGCGTGCTGGG CTACCAGAAGACCGTGGACCACACCAAGGTGAACAGCAAGCTGAGCCTGTTC TTCGAGATCAAGAGC (SEQ ID NO: 399) 3 MUMPSGTYRLIPNARANL GGCACCTACAGGCTGATCCCCAACGC IMMUNOE TA (SEQ ID NO:CAGGGCCAACCTGACCGCC (SEQ ID PITOPE 1 400) NO: 401) 4 MUMPSPSKFFTISDSATFA CCGAGCAAATTTTTTACCATTAGCGAT IMMUNOE PGPVSNA (SEQ IDAGCGCGACCTTTGCGCCGGGCCCGGT PITOPE 2 NO: 402) GAGCAACGCG (SEQ ID NO: 403)MUMPS PSKLFIMLDNATF CCGAGCAAACTGTTTATTATGCTGGAT IMMUNOE APGPVVNA (SEQAACGCGACCTTTGCGCCGGGCCCGGT PITOPE 1 ID NO: 404)GGTGAACGCG (SEQ ID NO: 405) SELECTED EXAMPLES FROMHEMAGGLUTININ-NEURAMINIDASE (HN) DIVERSITY AMONG OUTBREAKSTRAINS (TABLE 1) DIVERGENCE BETWEEN VACCINE STRAIN JL5 ANDOUTBREAK STRAINS (TABLE 2) 5 RUBELLA TPPPYQVSCGGESACCCCCCCCCCCTACCAGGTGAGCTGC VIRUS DRASARVIDPAAQGGCGGCGAGAGCGACAGGGCCAGCG (RV) S (SEQ ID NO: 406)CCAGGGTGATCGACCCCGCCGCCCAG AGC (SEQ ID NO: 407) 6 HEMAGGLUTININPEYAYKIVKNKK CCCGAGTACGCCTACAAGATCGTGAA MEDGFLQGMVDGAACAAGAAGATGGAGGACGGCTTCC GWYGHHSNEQGS TGCAGGGCATGGTGGACGGCTGGTACGLMENERTLDKA GGCCACCACAGCAACGAGCAGGGCAG NPNNDLCSWSDHCGGCCTGATGGAGAACGAGAGGACCC EASSNNTNQEDLL TGGACAAGGCCAACCCCAACAACGACQRESRRKKRIGTS CTGTGCAGCTGGAGCGACCACGAGGC TLNQRGNCNTKCCAGCAGCAACAACACCAACCAGGAGG QTEEARLKREEVS ACCTGCTGCAGAGGGAGAGCAGGAGGLVKSDQCSNGSLQ AAGAAGAGGATCGGCACCAGCACCCT CRANNSTEQVDGAACCAGAGGGGCAACTGCAACACCA (SEQ ID NO: 408) AGTGCCAGACCGAGGAGGCCAGGCTGAAGAGGGAGGAGGTGAGCCTGGTGA AGAGCGACCAGTGCAGCAACGGCAGCCTGCAGTGCAGGGCCAACAACAGCAC CGAGCAGGTGGAC (SEQ ID NO: 409) 7 NEURAMITKSTNSRSGGISG ACCAAGAGCACCAACAGCAGGAGCG NIDASE PDNEAPVGEAPSPGCGGCATCAGCGGCCCCGACAACGAG YGDNPRPNDGNN GCCCCCGTGGGCGAGGCCCCCAGCCCIRIGSKGYNGIITD CTACGGCGACAACCCCAGGCCCAACG TIEESCSCYPDAKACGGCAACAACATCAGGATCGGCAGC VVKSVELDSTIWT AAGGGCTACAACGGCATCATCACCGASGSSPNQKIITIGW CACCATCGAGGAGAGCTGCAGCTGCT DPNGWTGTPMSPACCCCGACGCCAAGGTGGTGAAGAGC NGAYGTDGPSNG GTGGAGCTGGACAGCACCATCTGGACQANQHQAESISA CAGCGGCAGCAGCCCCAACCAGAAGA GNSSLCPIRDNWHTCATCACCATCGGCTGGGACCCCAACG GSNRSWSWPDGA GCTGGACCGGCACCCCCATGAGCCCCE (SEQ ID NO: 410) AACGGCGCCTACGGCACCGACGGCCC CAGCAACGGCCAGGCCAACCAGCACCAGGCCGAGAGCATCAGCGCCGGCAAC AGCAGCCTGTGCCCCATCAGGGACAACTGGCACGGCAGCAACAGGAGCTGGA GCTGGCCCGACGGCGCCGAG (SEQ ID NO: 411) 8 TAP1EKIVLLLAMMEKI GAGAAGATCGTGCTGCTGCTGGCCATG (TRANSPORT VLLLAKCQTPMGATGGAGAAGATCGTGCTGCTGCTGGCC ANTIGEN AIKAVDGVTNKCPAAGTGCCAGACCCCCATGGGCGCCAT PRESENTATION) YLGSPSF (SEQ IDCAAGGCCGTGGACGGCGTGACCAACA ON NO: 412) AGTGCCCCTACCTGGGCAGCCCCAGC H5N1TTC (SEQ ID NO: 413) VIRUS HEMAGGLUTININ 9 TAP2 IRPCFWVELNPNQATCAGGCCCTGCTTCTGGGTGGAGCTG (TRANSPORT KIITIRPCFWVELIAACCCCAACCAGAAGATCATCACCATC ANTIGEN CYPDAGEIT(SEQAGGCCCTGCTTCTGGGTGGAGCTGATC PRESENTATION) ID NO: 414)TGCTACCCCGACGCCGGCGAGATCAC ON C (SEQ ID NO: 415) H5N1 VIRUSNEURAMINIDASE 10 HEMAGGLUTININ MEKIVLLLAEKIV ATGGAGAAGATCGTGCTGCTGCTGGCCEPITOPES LLLAMCPYLGSPS GAGAAGATCGTGCTGCTGCTGGCCATG TOWARD FKCQTPMGAIKAVTGCCCCTACCTGGGCAGCCCCAGCTTC CLASS I DGVTNK (SEQ IDAAGTGCCAGACCCCCATGGGCGCCAT HLA NO: 416) CAAGGCCGTGGACGGCGTGACCAACAAG (SEQ ID NO: 417) 11 NEURAMINIDASE NPNQKIITICYPDAGAACCCCAACCAGAAGATCATCACCATCT EITIRPCFWVELRPCGCTACCCCGACGCCGGCGAGATCACCAT EPITOPES FWVELI (SEQ IDCAGGCCCTGCTTCTGGGTGGAGCTGAG TOWARD NO: 418) GCCCTGCTTCTGGGTGGAGCTGATCCLASS I (SEQ ID NO: 419) HLA 12 HEMAGGLUTININ MVSLVKSDQIGTSTATGGTGAGCCTGGTGAAGAGCGACCAG EPITOPES LNQR (SEQ ID NO:ATCGGCACCAGCACCCTGAACCAGAGG TOWARD 420) (SEQ ID NO: 421) CLASS II HLA 13NEURAMINIDASE YNGIITDTI (SEQ ID TACAACGGCATCATCACCGACACCATC NO: 422)(SEQ ID NO: 423) EPITOPES TOWARD CLASS II HLA 14 HEMAGGLUTININMEKIVLLLAEKIVL ATGGAGAAGATCGTGCTGCTGCTGGCC EPITOPE LLAMMVSLVKSDQGAGAAGATCGTGCTGCTGCTGGCCATG H5N1- CPYLGSPSFIGTSTLATGGTGAGCCTGGTGAAGAGCGACCAG BOUND NQRKCQTPMGAIKTGCCCCTACCTGGGCAGCCCCAGCTTCA CLASS I AVDGVTNK(SEQTCGGCACCAGCACCCTGAACCAGAGG AND ID NO: 424) (SEQ ID NO: 425) CLASS II HLA15 NEURAMINIDASE NPNQKIITIYNGIIT AACCCCAACCAGAAGATCATCACCATCT EPITOPEDTICYPDAGEITIRP ACAACGGCATCATCACCGACACCATCTG H5N1- CFWVELRPCFWVECTACCCCGACGCCGGCGAGATCACCATC BOUND LI (SEQ ID NO: 426)AGGCCCTGCTTCTGGGTGGAGCTGAGG CLASS I CCCTGCTTCTGGGTGGAGCTGATC (SEQ ANDID NO: 427) CLASS II HLA

Additional immunoepitopes may also be installed which are known in theart. Any of the immunoepitopes available from the Immune EpitopeDatabase and Analysis Resource (iedb.org/epitopedetails_v3.php) (thecontents of which are incorporated herein by reference) may be installedby the prime editors disclosed herein.

In some embodiments, the immunoepitopes which may be installed by theprime editors disclosed herein may include any of the followingepitopes:

SEQ ID ACCESS. NO. DISEASE EPITOPE AMINO ACID SEQUENCE NO NO. 1 TETANUSQYIKANSKFIGITEL 396 NA TOXOID 2 DIPHTHERIA GADDVVDSSKSFVMENFSSYHGTKP 428NA TOXIN MUTANT GYVDSIQKGIQKPKSGTQGNYDDDW CRM197KEFYSTDNKYDAAGYSVDNENPLSG KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKR FGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDA MYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKN KMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYA AWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQS IALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKT QPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGK LDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAF HRSSSEKIHSNEISSDSIGVLGYQKTV DHTKVNSKLSLFFEIKS3 MUMPS GTYRLIPNARANLTA 400 NA 4 MUMPS PSKFFTISDSATFAPGPVSNA; 402; 404NA PSKLFIMLDNATFAPGPVVNA 5 RUBELLA VIRUS TPPPYQVSCGGESDRASARVIDPAAQ 406NA (RV) S 6 HEMAGGLUTININ PEYAYKIVKNKKMEDGFLQGMVDG 408 NAWYGHHSNEQGSGLMENERTLDKAN PNNDLCSWSDHEASSNNTNQEDLLQRESRRKKRIGTSTLNQRGNCNTKCQT EEARLKREEVSLVKSDQCSNGSLQCR ANNSTEQVD 7NEURAMINIDASE TKSTNSRSGGISGPDNEAPVGEAPSP 410 NAYGDNPRPNDGNNIRIGSKGYNGIITD TIEESCSCYPDAKVVKSVELDSTIWTSGSSPNQKIITIGWDPNGWTGTPMSPN GAYGTDGPSNGQANQHQAESISAGNSSLCPIRDNWHGSNRSWSWPDGAE 8 TAP (TRANSPORT EKIVLLLAMMEKIVLLLAKCQTPMG 412NA ANTIGEN AIKAVDGVTNKCPYLGSPSF PRESENTATION) ON H5N1 VIRUSHEMAGGLUTININ 9 TAP (TRANSPORT IRPCFWVELNPNQKIITIRPCFWVELIC 414 NAANTIGEN YPDAGEIT PRESENTATION) ON H5N1 VIRUS NEURAMINIDASE 10HEMAGGLUTININ MEKIVLLLAEKIVLLLAMCPYLGSPS 416 NA EPITOPESFKCQTPMGAIKAVDGVTNK TOWARD CLASS I HLA 11 NEURAMINIDASENPNQKIITICYPDAGEITIRPCFWVELR 418 NA EPITOPES PCFWVELI TOWARD CLASS I HLA12 HEMAGGLUTININ MVSLVKSDQIGTSTLNQR 420 NA EPITOPES TOWARD CLASS II HLA13 NEURAMINIDASE YNGIITDTI 422 NA EPITOPES TOWARD CLASS II HLA 14HEMAGGLUTININ MEKIVLLLAEKIVLLLAMMVSLVKSD 424 NA EPITOPE H5N1-QCPYLGSPSFIGTSTLNQRKCQTPMG BOUND CLASS I AIKAVDGVTNK AND CLASS II HLA 15NEURAMINIDASE NPNQKIITIYNGIITDTICYPDAGEITIR 426 NA EPITOPE H5N1-PCFWVELRPCFWVELI BOUND CLASS I AND CLASS II HLA 16 CORYNEBACTERIUMAACAGNRVRRSVGSSLKC 899 SRC280292 DIPHTHERIAE 17 MEASLES VIRUSAADHCPVVEVNGVTI 900 P69353.1 STRAIN EDMONSTON 18 MEASLES VIRUSAAHLPTGTPLDID 901 P04851.1 STRAIN EDMONSTON 19 BORDETELLA AALAVWAGLAVQ902 Q00879.1 PERTUSSIS 20 MEASLES VIRUS AALGVATAAQITAGI 903 P69353.1STRAIN EDMONSTON 21 RUBELLA VIRUS AALLNTPPPYQVSCGGESDRATAR 904 P07566.1STRAIN THERIEN 22 RUBELLA VIRUS AAQSFTGVVYGTHTT 905 BAA28178.1 23RUBELLA VIRUS ACEVEPAFGHSDAAC 906 BAA28178.1 24 RUBELLA VIRUSACTFWAVNAYSSGGY 907 BAA28178.1 25 RUBELLA VIRUS ADDPLLR 908 BAA19893.126 RUBELLA VIRUS ADDPLLRT 909 CAJ88851.1 27 MEASLES VIRUSAEMICDIDTYIVEAG 910 P04851.1 STRAIN EDMONSTON 28 MEASLES VIRUSAEMICDIDTYIVEAGLASFI 911 P04851.1 STRAIN EDMONSTON 29 MEASLES VIRUSAEPLLSC 912 P04851.1 STRAIN EDMONSTON 30 BORDETELLA AFVSTSSSRRYTEVY 913CAD44970.1 PERTUSSIS 31 BORDETELLA AGFIYRETFCITTIYKTGQPAADHYYS 914P04979.1 PERTUSSIS KVTA 32 RUBELLA VIRUS AGLLACCAKCLYYLR 915 BAA28178.133 RUBELLA VIRUS AHTTSDPWHPPG 916 BAA19893.1 34 MEASLES VIRUSAIAKLEDAKELLESS 917 P69353.1 STRAIN EDMONSTON 35 MEASLES VIRUSAIDNLRASLETTNQA 918 P69353.1 STRAIN EDMONSTON 36 BORDETELLA AKGVEFR 919ACI16088.1 PERTUSSIS 37 MEASLES VIRUS AKWAVPTTRTDDKLR 920 P08362.1STRAIN EDMONSTON 38 MEASLES VIRUS ALAEVLKKPV 921 ABO69699.1 STRAINEDMONSTON 39 MEASLES VIRUS ALGVINTLEWIPRFK 922 P08362.1 STRAIN EDMONSTON40 MEASLES VIRUS ALHQSMLNSQAIDNL 923 P69353.1 STRAIN EDMONSTON 41MEASLES VIRUS ALIGILSLFV 924 ABI54110.1 STRAIN EDMONSTON 42RUBELLA VIRUS ALLNTPPPYQVSCGGESDRA 925 CAJ88851.1 43 RUBELLA VIRUSALLNTPPPYQVSCGGESDRASARV 926 CAJ88851.1 STRAIN M33 44 RUBELLA VIRUSALVEGLAPGGGNCHL 927 BAA28178.1 45 BORDETELLA AMAAWSERAGEA 928 P04977.1PERTUSSIS 46 MEASLES VIRUS ANCASILCKCYTTGT 929 P69353.1 STRAIN EDMONSTON47 BORDETELLA ANPNPYTSRRSV 930 P04977.1 PERTUSSIS 48 RUBELLA VIRUSAPGPGEVW 931 CAJ88851.1 49 RUBELLA VIRUS APLPPHTTERIETRSARHPWRIR 932ABD64214.1 50 RUBELLA VIRUS APPMPPQPPRAHGQHYGHHHHQLPF 933 CAA33016.1VACCINE STRAIN LG RA27/3 51 RUBELLA VIRUS APPTLPQPPCAHGQHYGHHHHQLPF 934P07566.1 STRAIN THERIEN LG 52 RUBELLA VIRUS APPTLPQPPRAHGQHYGHHHHQLPF935 ABD64214.1 LG 53 BORDETELLA APQPGPQPPQPPQPQPEAPAPQ 936 P14283.3PERTUSSIS 54 RUBELLA VIRUS AQLASYFNPGGSYYK 937 BAA28178.1 55MEASLES VIRUS ARAAHLPTGTPLD 938 P04851.1 STRAIN EDMONSTON 56MEASLES VIRUS ARLVSEIAMHTTEDK 939 P04851.1 STRAIN EDMONSTON 57MEASLES VIRUS ARLVSEIAMHTTEDKISRAV 940 P04851.1 STRAIN EDMONSTON 58MEASLES VIRUS ASDVETAEGGEIHELLR 941 P03422.1 STRAIN EDMONSTON-B 59MEASLES ASDVETAEGGEIHELLRLQ 942 ABO69699.1 MORBILLIVIRUS 60MEASLES VIRUS ASDVETAEGGEIHELLRLQSR 943 P03422.1 STRAIN EDMONSTON-B 61MEASLES ASDVETAEGGEIHKLLRLQ 944 AAA63285.1 MORBILLIVIRUS 62RUBELLA VIRUS ASDVLPGHWLQG 945 NP_740663.1 STRAIN M33 63 MEASLES VIRUSASELGITAEDARLVS 946 P04851.1 STRAIN EDMONSTON 64 MEASLES VIRUSASELGITAEDARLVSEIAMH 947 P04851.1 STRAIN EDMONSTON 65 MEASLES VIRUSASESSQDPQDSRR 948 P04851.1 STRAIN EDMONSTON 66 MEASLES VIRUSASILCKCYTTGTIIN 949 P69353.1 STRAIN EDMONSTON 67 RUBELLA VIRUSASPVCQRHSPDCSRL 950 BAA28178.1 68 BORDETELLA ASQARWTGATRA 951 BAF35031.1PERTUSSIS 69 RUBELLA VIRUS ASYFNPGGSYYKQYHPTACEVEPAFG 952 P07566.1STRAIN THERIEN HS 70 MEASLES VIRUS ASYKVMTRSSHQSLV 953 P69353.1 STRAINEDMONSTON 71 BORDETELLA ASYVKKPKEDVD 954 ACI16088.1 PERTUSSIS 72MEASLES VIRUS ATAAQITAGIALHQS 955 P69353.1 STRAIN EDMONSTON 73RUBELLA VIRUS ATPERPRL 956 CAJ88851.1 74 MEASLES VIRUS AVCLGGLIGIPALIC957 P69353.1 STRAIN EDMONSTON 75 MEASLES VIRUS AVGPRQAQVSF 958 P04851.1STRAIN EDMONSTON 76 RUBELLA VIRUS AVNAYSSGGYAQLAS 959 BAA28178.1 77RUBELLA VIRUS AVSETRQTWAEWAAA 960 BAA28178.1 78 MEASLES VIRUSAVTAPDTAADSELRR 961 P04851.1 STRAIN EDMONSTON 79 MEASLES VIRUSAVTAPDTAADSELRRWIKYT 962 P04851.1 STRAIN EDMONSTON 80 BORDETELLAAYGGIIKDAPPGAGFIYRETFC 963 P04979.1 PERTUSSIS 81 RUBELLA VIRUSCALPLAGLLACCAKC 964 BAA28178.1 82 RUBELLA VIRUS CARIWNGTQRACTFW 965BAA28178.1 83 MEASLES VIRUS CARTLVSGSFGNRFI 966 P69353.1 STRAINEDMONSTON 84 BORDETELLA CASPYEGRYRDMYDALRBRLLY 967 SRC280066 PERTUSSIS85 BORDETELLA CAVFVRSGQPVIGA 968 AAA83981.1 PERTUSSIS 86 RUBELLA VIRUSCCAKCLYYLRGAIAPR 969 BAA28178.1 87 MEASLES VIRUS CCRGRCNKKGEQVGM 970P69353.1 STRAIN EDMONSTON 88 RUBELLA VIRUS CEIPTDVSCEGLGAW 971BAA28178.1 89 BORDETELLA CFGKDLKRPGSSPMEV 972 P0A3R5.1 PERTUSSIS 90MEASLES CFQQACKGKIQALCE 973 P06830.1 MORBILLIVIRUS 91 MEASLESCFQQACKGKIQALCENPEWAPLKDN 974 AAR89413.1 MORBILLIVIRUS RIPS 92RUBELLA VIRUS CGGESDRASARVIDP 975 BAA28178.1 93 BORDETELLACITTIYKTGQPAADHYYSKVTA 976 P04979.1 PERTUSSIS 94 MEASLES CKGKIQALCENPEWA977 AAR89413.1 MORBILLIVIRUS 95 MEASLES VIRUS CKPWQESRKNKAQ 978 P04851.1STRAIN EDMONSTON 96 MEASLES VIRUS CNKKGEQVGMSRPGL 979 P69353.1 STRAINEDMONSTON 97 RUBELLA VIRUS CNVTTEHPFCNTPHG 980 BAA28178.1 98 BORDETELLACQVGSSNSAF 981 P04977.1 PERTUSSIS 99 MEASLES CSGPTTIRGQFS 982 P08362.1MORBILLIVIRUS 100 BORDETELLA CTSPYDGKYWSMYSRL 983 AAA83981.1 PERTUSSIS101 MEASLES VIRUS CVLADSESGGHITHS 984 P08362.1 STRAIN EDMONSTON 102MEASLES CYTTGTIINQDPDKILTYIAADHC 985 AAF02706.1 MORBILLIVIRUS 103RUBELLA VIRUS DADDPLLR 986 CAJ88851.1 104 MEASLES VIRUS DARAAHLPTGTPLDI987 P04851.1 STRAIN EDMONSTON 105 MEASLES VIRUS DARAAHLPTGTPLDIDTASE 988P04851.1 STRAIN EDMONSTON 106 MEASLES VIRUS DCHAPTYLPAEVDGD 989 P08362.1STRAIN EDMONSTON 107 RUBELLA VIRUS DCSRLVGATPERPRL 990 BAA28178.1 108MEASLES VIRUS DDKLRMETCFQQACK 991 P08362.1 STRAIN EDMONSTON 109RUBELLA VIRUS DDPLLRTA 992 CAJ88851.1 110 RUBELLA VIRUS DDPLLRTAPGPGEVW993 BAA28178.1 111 BORDETELLA DDPPATVYRYD 994 P04977.1 PERTUSSIS 112BORDETELLA DDPPATVYRYDSRPPED 995 CAD44970.1 PERTUSSIS 113 BORDETELLADDPPATVYRYDSRPPEDV 996 ACI04548.1 PERTUSSIS 114 MEASLES DEVGLRTPQRFTDLV997 P06830.1 MORBILLIVIRUS 115 MEASLES VIRUS DHCPVVEVNGVTIQV 998P69353.1 STRAIN EDMONSTON 116 MEASLES VIRUS DIDTASESSQDPQ 999 P04851.1STRAIN EDMONSTON 117 MEASLES VIRUS DINKVLEKLGYSGGD 1000 P69353.1 STRAINEDMONSTON 118 BORDETELLA DLIAYKQ 1001 ACI16088.1 PERTUSSIS 119MEASLES VIRUS DLIGQKLGLKLLRYY 1002 P69353.1 STRAIN EDMONSTON 120RUBELLA VIRUS DLQKALEAQSRALRAELAA 1003 P07566.1 121 MEASLESDLQYVLATYDTSRVE 1004 P06830.1 MORBILLIVIRUS 122 MEASLES VIRUS DLSLRRFMV1005 P04851.1 STRAIN EDMONSTON 123 MEASLES VIRUS DLSNCMVALGELKLA 1006P08362.1 STRAIN EDMONSTON 124 RUBELLA VIRUS DLVEYIMNYTGNQQSRWGLGSPNC1007 CAJ88851.1 125 MEASLES DLVKFISDKIKFLNP 1008 AAR89413.1MORBILLIVIRUS 126 MEASLES DLVKFISTKIKFLNP 1009 SRC280117 MORBILLIVIRUS127 MEASLES VIRUS DLYKSNHNNV 1010 P08362.1 STRAIN EDMONSTON 128BORDETELLA DNVLDHLTGR 1011 ACI04548.1 PERTUSSIS 129 BORDETELLADNVLDHLTGRSC 1012 P04977.1 PERTUSSIS 130 BORDETELLA DNVLDHLTGRSCQ 1013P04977.1 PERTUSSIS 131 MEASLES DPDKILTYIAA 1014 AAF02706.1 MORBILLIVIRUS132 RUBELLA VIRUS DPGDLVEYIMNYTGNQQSR 1015 P07566.1 STRAIN THERIEN 133RUBELLA VIRUS DPLLRTAP 1016 CAJ88851.1 134 RUBELLA VIRUSDPLLRTAPGPGEVWVTPVIGSQ 1017 CAJ88851.1 135 MEASLES VIRUS DPQDSRRSAEPLL1018 P04851.1 STRAIN EDMONSTON 136 MEASLES VIRUS DPVIDRLYLSSHRGV 1019P08362.1 STRAIN EDMONSTON 137 MEASLES VIRUS DQILRSMKGLSSTSI 1020P69353.1 STRAIN EDMONSTON 138 MEASLES DQYCADVAAEELMNA 1021 P06830.1MORBILLIVIRUS 139 MEASLES VIRUS DSESGGHITH 1022 P08362.1 STRAINEDMONSTON 140 MEASLES VIRUS DTASESSQDPQDS 1023 P04851.1 STRAIN EDMONSTON141 RUBELLA VIRUS DTVMSVFALASYVQH 1024 BAA28178.1 142 BORDETELLADVFQNGFTAWGNND 1025 P04977.1 PERTUSSIS 143 RUBELLA VIRUS DVGAVPPGKFVTAAL1026 BAA28178.1 144 CORYNEBACTERIUM DVNKSKTHISVNGRKI 1027 CAE11230.1DIPHTHERIAE 145 RUBELLA VIRUS DVSCEGLGAWVPAAP 1028 BAA28178.1 146RUBELLA VIRUS DWASPVCQRHSPDCSRLVGATPERPR 1029 P07566.1 STRAIN THERIEN L147 MEASLES VIRUS EARESYRETGPSR 1030 P04851.1 STRAIN EDMONSTON 148BORDETELLA EAVEAERAGRGTG 1031 ACI04548.1 PERTUSSIS 149 MEASLES VIRUSEDAKELLESSDQILR 1032 P69353.1 STRAIN EDMONSTON 150 MEASLES VIRUSEDRRVKQSRGEAR 1033 P04851.1 STRAIN EDMONSTON 151 MEASLES VIRUSEDSITIPYQGSGKGV 1034 P08362.1 STRAIN EDMONSTON 152 RUBELLA VIRUSEEAFTYLCTAPGCAT 1035 BAA28178.1 153 MEASLES VIRUS EGFNMILGTILAQIW 1036P04851.1 STRAIN EDMONSTON 154 MEASLES VIRUS EGFNMILGTILAQIWVLLAK 1037P04851.1 STRAIN EDMONSTON 155 RUBELLA VIRUS EHPFCNTPHGQLEVQ 1038BAA28178.1 156 MEASLES VIRUS EISDIEVQDPEGFNM 1039 P04851.1 STRAINEDMONSTON 157 MEASLES VIRUS EISDIEVQDPEGFNMILGTI 1040 P04851.1 STRAINEDMONSTON 158 MEASLES VIRUS EKPNLSSKRSE 1041 P08362.1 STRAIN EDMONSTON159 MEASLES VIRUS ELKLAALCHGEDSIT 1042 P08362.1 STRAIN EDMONSTON 160MEASLES ELMNALVNSTLLETR 1043 P06830.1 MORBILLIVIRUS 161 MEASLES VIRUSELPRL 1044 P04851.1 STRAIN EDMONSTON 162 MEASLES ENPEWAPLKDNRIPSYGVLSVDL1045 AAR89413.1 MORBILLIVIRUS 163 MEASLES VIRUS EPIRDALNAMTQNIR 1046P69353.1 STRAIN EDMONSTON 164 MEASLES VIRUS EQVGMSRPGLKPDLT 1047P69353.1 STRAIN EDMONSTON 165 RUBELLA VIRUS ERPRLRLV 1048 CAJ88851.1 166RUBELLA VIRUS ERPRLRLVDADDPLL 1049 BAA28178.1 167 MEASLES VIRUSESPGQLIQRITDDPDVS 1050 P04851.1 CAM/RB 168 MEASLES VIRUS ESRGIKARITHVDTE1051 P69353.1 STRAIN EDMONSTON 169 MEASLES VIRUS ESSCTFMPEGTVCSQ 1052P69353.1 STRAIN EDMONSTON 170 MEASLES VIRUS ESSQDPQDSRRSA 1053 P04851.1STRAIN EDMONSTON 171 MEASLES VIRUS ETRTTNQFLAVSKGN 1054 P08362.1 STRAINEDMONSTON 172 MEASLES VIRUS EVDGDVKLSSNLVIL 1055 P08362.1 STRAINEDMONSTON 173 MEASLES VIRUS EVNGVTIQV 1056 P26031.1 STRAIN EDMONSTON-B174 RUBELLA VIRUS EVWVTPVI 1057 CAJ88851.1 175 RUBELLA VIRUSEVWVTPVIGSQA 1058 BAA19893.1 176 RUBELLA VIRUS EWAAAHWWQLTLGAT 1059BAA28178.1 177 MEASLES EWIPRFKVSPYLFTV 1060 P06830.1 MORBILLIVIRUS 178BORDETELLA FEYVDTYGDNAG 1061 P04977.1 PERTUSSIS 179 MEASLESFGPLITHGSGMDLYK 1062 P06830.1 MORBILLIVIRUS 180 MEASLES VIRUS FIFDALAEV1063 ABK40531.1 STRAIN EDMONSTON 181 MEASLES VIRUS FISDKIKFL 1064P08362.1 STRAIN EDMONSTON 182 MEASLES VIRUS FKRNKDKPPITSGSG 1065P04851.1 STRAIN EDMONSTON 183 MEASLES VIRUS FKRNKDKPPITSGSGGAIRG 1066P04851.1 STRAIN EDMONSTON 184 RUBELLA VIRUS FKTVRPVALPRTLAP 1067BAA28178.1 185 MEASLES VIRUS FLMDRHIIV 1068 ABK40531.1 STRAIN EDMONSTON186 MEASLES VIRUS FMAVLLTLQTPTGQI 1069 P69353.1 STRAIN EDMONSTON 187MEASLES VIRUS FMPEGTVCSQNALYP 1070 P69353.1 STRAIN EDMONSTON 188 MEASLESFMYMSLLGV 1071 AAN09804.1 MORBILLIVIRUS 189 MEASLES VIRUSFNVPIKEAGEDCHAP 1072 P08362.1 STRAIN EDMONSTON 190 MEASLESFRDLTWCINPPERIK 1073 AAC35876.2 MORBILLIVIRUS 191 MEASLES VIRUSFSHDDPISSDQSRFG 1074 P04851.1 STRAIN EDMONSTON 192 MEASLES VIRUSFSHDDPISSDQSRFGWFENK 1075 P04851.1 STRAIN EDMONSTON 193 MEASLESFTDLVKFISDKIKFL 1076 P06830.1 MORBILLIVIRUS 194 MEASLES FTWDQKLWCRHFCVL1077 P06830.1 MORBILLIVIRUS 195 BORDETELLA FVRDGQSVIGACASPYEGRYRDLYD1078 SRC280066 PERTUSSIS ALRRLLY 196 BORDETELLAFVRSGQPVIGACTSPYDGKYWSILYS 1079 SRC280066 PERTUSSIS RLRKMLY 197 MEASLESFYKDNPHPKGSRIVI 1080 P06830.1 MORBILLIVIRUS 198 BORDETELLAGAASSYFEYVDTYG 1081 ACI04548.1 PERTUSSIS 199 BORDETELLAGAFDLKTTFCIMTTRNTGQPA 1082 AAA83981.1 PERTUSSIS 200 MEASLES VIRUSGALIGILSLFVESPG 1083 P04851.1 STRAIN EDMONSTON 201 MEASLES VIRUSGALIGILSLFVESPGQLIQR 1084 P04851.1 STRAIN EDMONSTON 202 RUBELLA VIRUSGATPERPR 1085 CAJ88851.1 203 BORDETELLA GAYGRCPNGTRALTVAELRGNAEL 1086P04979.1 PERTUSSIS 204 RUBELLA VIRUS GCFAPWDLEATGACI 1087 BAA28178.1 205MEASLES GDINKVLEKLGYS 1088 BAB60865.1 MORBILLIVIRUS 206 MEASLESGDINKVLEKLGYSGGDLLG 1089 AAL29688.1 MORBILLIVIRUS 207 RUBELLA VIRUSGDLRAVHHRPVPA 1090 CAA28880.1 208 RUBELLA VIRUS GDLVEYIMNYTGNQQ 1091BAA28178.1 209 MEASLES VIRUS GDSSITTRSRLLDRL 1092 P04851.1 STRAINEDMONSTON 210 MEASLES VIRUS GDSSITTRSRLLDRLVRLIG 1093 P04851.1 STRAINEDMONSTON 211 MEASLES GEDCHAPTYLPAEVD 1094 P06830.1 MORBILLIVIRUS 212MEASLES VIRUS GELSTLESLMNLYQQ 1095 P04851.1 STRAIN EDMONSTON 213MEASLES VIRUS GELSTLESLMNLYQQMGKPA 1096 P04851.1 STRAIN EDMONSTON 214MUMPS GEQARYLALLEA 1097 P21186.1 RUBULAVIRUS 215 RUBELLA VIRUS GEVWVT1098 BAA19893.1 216 RUBELLA VIRUS GEVWVTPV 1099 CAJ88851.1 217RUBELLA VIRUS GEVWVTPVIGSQAR 1100 BAA19893.1 218 BORDETELLAGEYGGVIKDGTPGGA 1101 AAA83981.1 PERTUSSIS 219 RUBELLA VIRUSGFLSGVGPMRLRHGADT 1102 SRC265968 220 MEASLES VIRUSGFRASDVETAEGGEIHELLRLQ 1103 P03422.1 STRAIN EDMONSTON-B 221 BORDETELLAGGAVPGGAVPGGAVPGGFGPGGFGP 1104 P14283.3 PERTUSSIS 222 BORDETELLAGGAVPGGAVPGGFGPGGFGPGGFGP 1105 CAA09475.1 PERTUSSIS 223 BORDETELLAGGAVPGGAVPGGFGPGGFGPGGFGP 1106 CAA09474.1 PERTUSSIS GGFGP 224 MEASLESGGHITHSGMVGMGVS 1107 P06830.1 MORBILLIVIRUS 225 MEASLESGILESRGIKARITHVDTESY 1108 P26032.1 MORBILLIVIRUS 226 BORDETELLAGITGETTTTEYSNARYV 1109 CAD44970.1 PERTUSSIS 227 MEASLES VIRUSGKEDRRVKQSRGE 1110 P04851.1 STRAIN EDMONSTON 228 BORDETELLA GKVTNGS 1111ACI16088.1 PERTUSSIS 229 RUBELLA VIRUS GLGAWVPAAPCARIW 1112 BAA28178.1230 MEASLES VIRUS GLIGIPALICCCRGR 1113 P69353.1 STRAIN EDMONSTON 231RUBELLA VIRUS GLLACCAKCLYYLRGAIAPR 1114 P07566.1 STRAIN THERIEN 232MEASLES GLLAIAGIRLHRAAI 1115 P06830.1 MORBILLIVIRUS 233 RUBELLA VIRUSGLQPRADMAAPPTLPQ 1116 NP_740663.1 234 MEASLES GMGVSCTVTREDGTNRR 1117AAR89413.1 MORBILLIVIRUS 235 MEASLES GMYGGTYLVEKP 1118 AAR89413.1MORBILLIVIRUS 236 BORDETELLA GNAELQTYLRQITPGWSIYGLYDGTY 1119 P04979.1PERTUSSIS LG 237 RUBELLA VIRUS GNCHLTVNGEDVGAV 1120 BAA28178.1 238BORDETELLA GNNDNVLDHLTGR 1121 P04977.1 PERTUSSIS 239 BORDETELLAGNNDNVLDHLTGRSC 1122 P04977.1 PERTUSSIS 240 MEASLES VIRUSGNRFILSQGNLIANC 1123 P69353.1 STRAIN EDMONSTON 241 RUBELLA VIRUSGNRGRGQRRDWSRAPPPPEERQETR 1124 P07566.1 SQTPAPKPS 242 RUBELLA VIRUSGPGEVWVT 1125 CAJ88851.1 243 RUBELLA VIRUS GPMRLRHGADTRCGRLI 1126P07566.1 STRAIN THERIEN 244 BORDETELLA GPNHTKV 1127 ACI16083.1 PERTUSSIS245 MEASLES VIRUS GPRQAQVSF 1128 P10050.1 STRAIN HALLE 246 MEASLES VIRUSGPRQAQVSFLQGDQS 1129 P04851.1 STRAIN EDMONSTON 247 MEASLES VIRUSGPRQAQVSFLQGDQSENELP 1130 P04851.1 STRAIN EDMONSTON 248 MEASLESGRGYNVSSIVTMTSQ 1131 P06830.1 MORBILLIVIRUS 249 BORDETELLA GRTPFII 1132ACI16083.1 PERTUSSIS 250 RUBELLA VIRUS GSPNCHGPDWASPVC 1133 BAA28178.1251 RUBELLA VIRUS GSQARKCGLHIRAGP 1134 BAA28178.1 252 BORDETELLAGSSNSAFVSTSSSRR 1135 P04977.1 PERTUSSIS 253 MEASLES VIRUSGSTKSCARTLVSGSF 1136 P69353.1 STRAIN EDMONSTON 254 RUBELLA VIRUSGSYYKQYHPTACEVE 1137 BAA28178.1 255 RUBELLA VIRUS GTHTTAVSETRQTWA 1138BAA28178.1 256 MEASLES VIRUS GTIINQDPDKILTYI 1139 P69353.1 STRAINEDMONSTON 257 BORDETELLA GTLVRMAPVIG 1140 ADA85124.1 PERTUSSIS 258MEASLES VIRUS GTPLDIDTASESS 1141 P04851.1 STRAIN EDMONSTON 259BORDETELLA GTYLGQAYGGIIKDAPPGAGFIYRETF 1142 P04979.1 PERTUSSIS C 260BORDETELLA GVATKGLGVHAKSSDWG 1143 P15318.2 PERTUSSIS 261 CORYNEBACTERIUMGVLLPTIPGKLDVNKSKTHI 1144 AAV70486.1 DIPHTHERIAE 262 MEASLESGVLSVDLSLTVELKI 1145 P06830.1 MORBILLIVIRUS 263 MEASLES GVPIELQVECFTWDQ1146 P06830.1 MORBILLIVIRUS 264 MEASLES VIRUS GVSCTVTREDGTNRR 1147P08362.1 STRAIN EDMONSTON 265 MEASLES VIRUS GVSYNIGSQEWYTTV 1148P69353.1 STRAIN EDMONSTON 266 MEASLES VIRUS GYNVSSIVTMTSQGM 1149P08362.1 STRAIN EDMONSTON 267 MEASLES HFCVLADSESGGHIT 1150 P06830.1MORBILLIVIRUS 268 MEASLES HGEDSITIPYQGSGK 1151 P06830.1 MORBILLIVIRUS269 RUBELLA VIRUS HGPDWASP 1152 BAA19893.1 270 RUBELLA VIRUSHGPDWASPVCQRHSP 1153 BAA28178.1 271 RUBELLA VIRUS HGPDWASPVCQRHSPDCSRLVG1154 CAJ88851.1 272 RUBELLA VIRUS HGPDWASPVCQRHSPDCSRLVGATPE 1155CAJ88851.1 STRAIN M33 RPRLRLV 273 MEASLES VIRUS HITHSGMEGMGVSCT 1156P08362.1 STRAIN EDMONSTON 274 MEASLES HKSLSTNLDVTNSIE 1157 P06830.1MORBILLIVIRUS 275 MEASLES VIRUS HLMIDRPYV 1158 P08362.1 STRAIN EDMONSTON276 MEASLES VIRUS HLPTGTPLDIDTA 1159 P04851.1 STRAIN EDMONSTON 277MEASLES VIRUS HLPTGTPLDIDTATESSQDPQDSR 1160 Q77M43.1 STRAIN EDMONSTON-B278 MEASLES HMTNYLEQPVSNDLS 1161 P06830.1 MORBILLIVIRUS 279MEASLES VIRUS HQSLVIKLMPNITLL 1162 P69353.1 STRAIN EDMONSTON 280 MEASLESHRAAIYTAEIHKSLS 1163 P06830.1 MORBILLIVIRUS 281 BORDETELLAHRMQEAVEAERAGRGTGH 1164 P04977.1 PERTUSSIS 282 MEASLES VIRUSHVDTESYFIVLSIAY 1165 P69353.1 STRAIN EDMONSTON 283 MEASLES VIRUSHWGNLSKIGVVGIGS 1166 P69353.1 STRAIN EDMONSTON 284 RUBELLA VIRUSHWWQLTLGATCALPL 1167 BAA28178.1 285 RUBELLA VIRUSHYRNASDVLPGHWLQGGWGCYNL 1168 NP_740663.1 286 MEASLES VIRUSIDLGPPISLERLDVG 1169 P69353.1 STRAIN EDMONSTON 287 MEASLES VIRUSIEAIRQAGQEMILAV 1170 P69353.1 STRAIN EDMONSTON 288 RUBELLA VIRUSIETRSARHP 1171 CAA28880.1 STRAIN M33 289 MEASLES VIRUS IGSQEWYTTVPKYVA1172 P69353.1 STRAIN EDMONSTON 290 MEASLES VIRUS IKGVIVHRLEGVSYN 1173P69353.1 STRAIN EDMONSTON 291 MEASLES VIRUS IKHIIIVPIPGDSSI 1174P04851.1 STRAIN EDMONSTON 292 MEASLES VIRUS IKHIIIVPIPGDSSITTRSR 1175P04851.1 STRAIN EDMONSTON 293 BORDETELLA IKLKDCP 1176 ACI16083.1PERTUSSIS 294 MEASLES VIRUS IKLMPNITLLNNCTR 1177 P69353.1 STRAINEDMONSTON 295 MEASLES ILLERLDVGT 1178 AAF85664.1 MORBILLIVIRUS 296MEASLES ILPGQDLQYV 1179 P08362.1 MORBILLIVIRUS 297 MEASLES VIRUSILTYIAADHCPVVEV 1180 P69353.1 STRAIN EDMONSTON 298 MEASLES INQDPDKILTY1181 AAL29688.1 MORBILLIVIRUS 299 MEASLES VIRUS IPRFKVSPYLFNVPI 1182P08362.1 STRAIN EDMONSTON 300 MEASLES VIRUS IQALSYALGGDINKV 1183P69353.1 STRAIN EDMONSTON 301 RUBELLA VIRUS IRAGPYGHATVEMPE 1184BAA28178.1 302 BORDETELLA IRMGTDK 1185 ACI16088.1 PERTUSSIS 303MEASLES VIRUS ISNFDESSCTFMPEG 1186 P69353.1 STRAIN EDMONSTON 304MEASLES VIRUS ITAGIALHQSMLNSQ 1187 P69353.1 STRAIN EDMONSTON 305MEASLES VIRUS ITDDPDVSIRLLEVV 1188 P04851.1 STRAIN EDMONSTON 306MEASLES VIRUS ITDDPDVSIRLLEVVQSDQS 1189 P04851.1 STRAIN EDMONSTON 307BORDETELLA ITTYV 1190 ACI16083.1 PERTUSSIS 308 MEASLES VIRUSIVEAGLASFILTIKF 1191 P04851.1 STRAIN EDMONSTON 309 MEASLES VIRUSIVEAGLASFILTIKFGIETM 1192 P04851.1 STRAIN EDMONSTON 310 MEASLES VIRUSIVEAGLASFILTIKFGIETMYPALG 1193 P04851.1 STRAIN EDMONSTON 311RUBELLA VIRUS KALEAQSRALRAELAA 1194 P07566.1 312 MEASLES VIRUSKARITHVDTESYFIV 1195 P69353.1 STRAIN EDMONSTON 313 RUBELLA VIRUSKCGLHIRAGPYGHAT 1196 BAA28178.1 314 MEASLES VIRUS KCYTTGTIINQDPDK 1197P69353.1 STRAIN EDMONSTON 315 MEASLES VIRUS KDNPHPKGSR 1198 P08362.1STRAIN EDMONSTON 316 MEASLES VIRUS KDNRIPSYGVLSVDL 1199 P08362.1 STRAINEDMONSTON 317 MEASLES KFLNPDREYDFRDLT 1200 AAC35876.2 MORBILLIVIRUS 318RUBELLA VIRUS KFVTAALLN 1201 BAA28178.1 319 MEASLES VIRUS KGNCSGPTTIR1202 P08362.1 STRAIN EDMONSTON 320 MEASLES KIKFLNPDREYDFRD 1203 P06830.1MORBILLIVIRUS 321 RUBELLA VIRUS KIVDGGCFAPWDLEA 1204 BAA28178.1 322MEASLES VIRUS KLGLKLLRYYTEILS 1205 P69353.1 STRAIN EDMONSTON 323MEASLES VIRUS KLGVWKSPTDMQSWV 1206 P08362.1 STRAIN EDMONSTON 324BORDETELLA KLKECPQ 1207 ACI16088.1 PERTUSSIS 325 MEASLES KLLRYYTEI 1208P26031.1 MORBILLIVIRUS 326 MEASLES VIRUS KLMPFSGDFV 1209 ABK40531.1STRAIN EDMONSTON 327 MEASLES KLMPNITLL 1210 P26031.1 MORBILLIVIRUS 328MEASLES KLRMETCFQQACKGKIQALCENPEW 1211 AAR89413.1 MORBILLIVIRUS A 329MEASLES KLWCRHFCV 1212 P08362.1 MORBILLIVIRUS 330 MEASLES VIRUSKLWCRHFCVL 1213 P08362.1 STRAIN EDMONSTON 331 MEASLES KLWESPQEI 1214BAB60863.1 MORBILLIVIRUS 332 MEASLES VIRUS KMSSAVGFV 1215 ABO69699.1STRAIN EDMONSTON 333 MEASLES VIRUS KMSSAVGFVPDTGPASR 1216 P03422.1STRAIN EDMONSTON-B 334 BORDETELLA KMVYATN 1217 ACI16083.1 PERTUSSIS 335MEASLES VIRUS KPDLTGTSKSYVRSL 1218 P69353.1 STRAIN EDMONSTON 336 MEASLESKPNLSSKRSELSQLS 1219 P08362.1 MORBILLIVIRUS 337 MEASLES VIRUSKPNLSSKRSELSQLSMYRVF 1220 P08362.1 STRAIN EDMONSTON 338 MEASLES VIRUSKQSRGEARESYRETG 1221 P04851.1 STRAIN EDMONSTON 339 MEASLES VIRUSKQSRGEARESYRETGPSRAS 1222 P04851.1 STRAIN EDMONSTON 340 MEASLES VIRUSKRFAGVVLAGAALGV 1223 P69353.1 STRAIN EDMONSTON 341 MEASLES VIRUSKRTPGNKPRIAEMIC 1224 P04851.1 STRAIN EDMONSTON 342 MEASLES VIRUSKRTPGNKPRIAEMICDIDTY 1225 P04851.1 STRAIN EDMONSTON 343 MEASLESKSNHNNVYWLTIPPMKNLALGVINT 1226 AAR89413.1 MORBILLIVIRUS L 344 MEASLESKVSPYLFNV 1227 P08362.1 MORBILLIVIRUS 345 BORDETELLA KVVQLPKISKNALKANG1228 ACI16083.1 PERTUSSIS 346 BORDETELLA KVVQLPKISKNALRNDG 1229ACI16087.1 PERTUSSIS 347 BORDETELLA LAHRRIPPENIR 1230  P04977.1PERTUSSIS 348 BORDETELLA LALALWAGFALS 1231 P11092.1 PERTUSSIS 349RUBELLA VIRUS LAPGGGNCHLTVNGE 1232 BAA28178.1 350 MEASLES VIRUSLAQIWVLLAKAVTAP 1233 P04851.1 STRAIN EDMONSTON 351 MEASLES VIRUSLAQIWVLLAKAVTAPDTAAD 1234 P04851.1 STRAIN EDMONSTON 352 RUBELLA VIRUSLASYFNPGGSYYKQYHPTACEVEPAF 1235 BAA19893.1 GHS 353 MEASLESLAVSKGNCSGPTTIR 1236 P06830.1 MORBILLIVIRUS 354 MEASLES VIRUSLCENPEWAPLKDNRI 1237 P08362.1 STRAIN EDMONSTON 355 MEASLES LDRLVRLIG1238 ABI54110.1 MORBILLIVIRUS 356 CORYNEBACTERI LEEEGVTPL 1239 P33120.2DIPHTHERIAEUM 357 BORDETELLA LEHRMQEAVEAERAGRGTGHFI 1240 CAD44970.1PERTUSSIS 358 MEASLES VIRUS LEKLGYSGGDLLGIL 1241 P69353.1 STRAINEDMONSTON 359 MEASLES VIRUS LEQPVSNDLS 1242 P08362.1 STRAIN EDMONSTON360 MEASLES VIRUS LERKWLDVVRNIIAE 1243 P04851.1 STRAIN EDMONSTON 361MEASLES VIRUS LERKWLDVVRNIIAEDLSLR 1244 P04851.1 STRAIN EDMONSTON 362MEASLES VIRUS LFGPSLRDPISAEIS 1245 P69353.1 STRAIN EDMONSTON 363 MEASLESLGELKLAALCHGEDS 1246 P06830.1 MORBILLIVIRUS 364 MEASLES VIRUSLGGKEDRRVKQSR 1247 P04851.1 STRAIN EDMONSTON 365 RUBELLA VIRUSLGHDGHHGGTLRVGQHHRNASDVL 1248 ABD64214.1 366 RUBELLA VIRUSLGSPNCHGPDWASPVCQRHS 1249 P07566.1 STRAIN THERIEN 367 RUBELLA VIRUSLGSPNCHGPDWASPVCQRHSPDCSRL 1250 P07566.1 STRAIN THERIEN V 368RUBELLA VIRUS LHDPDTEAPTEACVTSWL 1251 ABD64214.1 369 MEASLES VIRUSLIANCASILCKCYTT 1252 P69353.1 STRAIN EDMONSTON 370 MEASLESLIGLLAIAGIRLHRAAIYTAEIHK 1253 AAR89413.1 MORBILLIVIRUS 371 MEASLES VIRUSLIPSMNQLSCDLIGQ 1254 P69353.1 STRAIN EDMONSTON 372 MEASLES VIRUSLKIKIASGFGPLITH 1255 P08362.1 STRAIN EDMONSTON 373 MEASLESLKIKIASGFGPLITHGSGMDLYK 1256 AAR89413.1 MORBILLIVIRUS 374 BORDETELLALKLYFEP 1257 ACI16088.1 PERTUSSIS 375 MEASLES VIRUS LLAVLFVMFL 1258P08362.1 STRAIN EDMONSTON 376 MEASLES VIRUS LLDRLVRLIGNPDVS 1259P04851.1 STRAIN EDMONSTON 377 MEASLES VIRUS LLDRLVRLIGNPDVSGPKLT 1260P04851.1 STRAIN EDMONSTON 378 MEASLES VIRUS LLESSDQILRSMKGL 1261P69353.1 STRAIN EDMONSTON 379 MEASLES LLETRTTNQFLAVSK 1262 P06830.1MORBILLIVIRUS 380 MEASLES VIRUS LLEVVQSDQSQSGLT 1263 P04851.1 STRAINEDMONSTON 381 MEASLES VIRUS LLEVVQSDQSQSGLTFASR 1264 P04851.1 STRAINEDMONSTON 382 MEASLES VIRUS LLEVVQSDQSQSGLTFASRG 1265 P04851.1 STRAINEDMONSTON 383 MEASLES LLGILESRGIKARIT 1266 AAL29688.1 MORBILLIVIRUS 384RUBELLA VIRUS LLRTAPGP 1267 CAJ88851.1 385 MEASLES VIRUS LLRYYTEILSLFGPS1268 P69353.1 STRAIN EDMONSTON 386 RUBELLA VIRUS LLVPWVLIFMVCRRACRRRG1269 P07566.1 STRAIN THERIEN 387 MEASLES VIRUS LLWRSRCKIV 1270ABK40528.1 STRAIN EDMONSTON 388 MEASLES LLWSYAMGV 1271 P04851.1MORBILLIVIRUS 389 MEASLES VIRUS LLWSYAMGVGVELEN 1272 P04851.1 STRAINEDMONSTON 390 MEASLES VIRUS LLWSYAMGVGVELENSMGGL 1273 P04851.1 STRAINEDMONSTON 391 MEASLES LMIDRPYVL 1274 P08362.1 MORBILLIVIRUS 392MEASLES VIRUS LNAMTQNIRPVQSVA 1275 P69353.1 STRAIN EDMONSTON 393RUBELLA VIRUS LNTPPPYQVSCGGES 1276 BAA28178.1 394 MEASLES VIRUSLRDPISAEISIQALS 1277 P69353.1 STRAIN EDMONSTON 395 BORDETELLALRGSGDLQEYLRHVTR 1278 AAA83981.1 PERTUSSIS 396 RUBELLA VIRUS LRLVDADD1279 CAJ88851.1 397 RUBELLA VIRUS LRLVDADDPLLR 1280 BAA19893.1 398RUBELLA VIRUS LRLVDADDPLLRTAPGPGEVWVTPVI 1281 BAA19893.1 GSQAR 399BORDETELLA LRRLLYMIYMSGLAVRVHVSKEEQY 1282 P04979.1 PERTUSSIS YDY 400RUBELLA VIRUS LRTAPGPG 1283 CAJ88851.1 401 RUBELLA VIRUSLRVGQHYRNASDVLPGHWLQ 1284 NP_740663.1 402 BORDETELLA LRYLA 1285ACI16088.1 PERTUSSIS 403 MEASLES VIRUS LSCKPWQESRKNK 1286 P04851.1STRAIN EDMONSTON 404 MEASLES LSEIKGVIVHRLEGV 1287 AAL29688.1MORBILLIVIRUS 405 MEASLES VIRUS LSIAYPTLSEIKGVI 1288 P69353.1 STRAINEDMONSTON 406 MEASLES VIRUS LSLLDLYLGRGYNVS 1289 P08362.1 STRAINEDMONSTON 407 MEASLES VIRUS LSQGNLIANCASILC 1290 P69353.1 STRAINEDMONSTON 408 MEASLES LSSHRGVIADNQAKW 1291 P06830.1 MORBILLIVIRUS 409MEASLES VIRUS LSVDLSLTVELKIKI 1292 P08362.1 STRAIN EDMONSTON 410BORDETELLA LTGISICNPGSSLC 1293 AAA83981.1 PERTUSSIS 411 MEASLES VIRUSLTIKFGIETMYPALG 1294 P04851.1 STRAIN EDMONSTON 412 MEASLES VIRUSLTIKFGIETMYPALGLHEFA 1295 P04851.1 STRAIN EDMONSTON 413 MEASLES VIRUSLTLQTPTGQIHWGNL 1296 P69353.1 STRAIN EDMONSTON 414 RUBELLA VIRUSLVDADDPL 1297 CAJ88851.1 415 RUBELLA VIRUS LVDADDPLLR 1298 BAA19893.1416 MEASLES VIRUS LVEKPNLSSKRSELS 1299 P08362.1 STRAIN EDMONSTON 417RUBELLA VIRUS LVGATPE 1300 BAA19893.1 418 RUBELLA VIRUS LVGATPER 1301CAJ88851.1 419 MEASLES LVKLGVWKSPTGMQS 1302 P06830.1 MORBILLIVIRUS 420MEASLES VIRUS LVSGSFGNRFILSQGNLI 1303 P26031.1 STRAIN EDMONSTON-B 421MEASLES VIRUS LYKSNHNNVYWLTIP 1304 P08362.1 STRAIN EDMONSTON 422MEASLES VIRUS LYPMSPLLQECLRGSTKSCARTLVS 1305 P69353.1 STRAIN EDMONSTON423 RUBELLA VIRUS MASTTPITMEDLQKALEA 1306 P07566.1 424 RUBELLA VIRUSMASTTPITMEDLQKALEAQSR 1307 ABD64200.1 425 RUBELLA VIRUSMASTTPITMEDLQKALEAQSRALRA 1308 P07566.1 STRAIN THERIEN ELAA 426RUBELLA VIRUS MASTTPITMEDLQKALEAQSRALRA 1309 ABD64200.1 GLAA 427RUBELLA VIRUS MASTTPITMEDLQKALETQSRVLRAG 1310 CAA33016.1 VACCINE STRAINLTA RA27/3 428 MEASLES VIRUS MATLLRSLALFKRNK 1311 P04851.1 STRAINEDMONSTON 429 MEASLES VIRUS MATLLRSLALFKRNKDKPPI 1312 P04851.1 STRAINEDMONSTON 430 MEASLES MDLYKSNHNNVYWLT 1313 P06830.1 MORBILLIVIRUS 431RUBELLA VIRUS MEDLQKALEAQSRA 1314 P07566.1 432 RUBELLA VIRUSMEDLQKALEAQSRALRAELAA 1315 P07566.1 433 MEASLES VIRUS MGLKVNVSAIFMAVL1316 P69353.1 STRAIN EDMONSTON 434 MEASLES MIDRPYVLLAVLFVM 1317 P06830.1MORBILLIVIRUS 435 MEASLES VIRUS MILAVQGVQDYINNE 1318 P69353.1 STRAINEDMONSTON 436 MEASLES VIRUS MLNSQAIDNLRASLE 1319 P69353.1 STRAINEDMONSTON 437 MEASLES VIRUS MNALVNSTLLETRTT 1320 P08362.1 STRAINEDMONSTON 438 RUBELLA VIRUS MNYTGNQQSRWGLGSPNCHGPDWA 1321 BAA19893.1SPVCQRHS 439 MEASLES VIRUS MQSWVPLSTDDPVID 1322 P08362.1 STRAINEDMONSTON 440 MEASLES MSLSLLDLYLGRGYN 1323 P06830.1 MORBILLIVIRUS 441MEASLES VIRUS MSPLLQECLRGSTKS 1324 P69353.1 STRAIN EDMONSTON 442 MEASLESMSPQRDRINAFYKDN 1325 P06830.1 MORBILLIVIRUS 443 MEASLES MYRVFEVSVIRNPGL1326 P06830.1 MORBILLIVIRUS 444 MEASLES VIRUS NALYPMSPLLQECLR 1327P69353.1 STRAIN EDMONSTON 445 RUBELLA VIRUS NCHGPDWASPVCQRHSPDCSRLVGA1328 P07566.1 STRAIN THERIEN T 446 MEASLES VIRUS NFGRSYFDPAYFRLG 1329P04851.1 STRAIN EDMONSTON 447 MEASLES VIRUS NFGRSYFDPAYFRLGQEMVR 1330P04851.1 STRAIN EDMONSTON 448 RUBELLA VIRUS NGTQRACTFWAVNAY 1331BAA28178.1 449 MEASLES VIRUS NGVTIQVGSRRYPDA 1332 P69353.1 STRAINEDMONSTON 450 MEASLES VIRUS NIIAEDLSLRRFMVA 1333 P04851.1 STRAINEDMONSTON 451 MEASLES VIRUS NIIAEDLSLRRFMVALILDI 1334 P04851.1 STRAINEDMONSTON 452 MEASLES VIRUS NITLLNNCTRVEIAE 1335 P69353.1 STRAINEDMONSTON 453 MEASLES NLALGVINTLEWIPR 1336 P06830.1 MORBILLIVIRUS 454CORYNEBACTERIUM NLFQVVHWSYNRPAYSPG 1337 SRC280292 DIPHTHERIAE 455MEASLES VIRUS NLVILPGQDLQYVLA 1338 P08362.1 STRAIN EDMONSTON 456MEASLES VIRUS NLYQQMGKPAPYMVN 1339 P04851.1 STRAIN EDMONSTON 457MEASLES VIRUS NLYQQMGKPAPYMVNLENSI 1340 P04851.1 STRAIN EDMONSTON 458MEASLES VIRUS NNCTRVEIAEYRRLL 1341 P69353.1 STRAIN EDMONSTON 459MEASLES VIRUS NPDREYDFRD 1342 P08362.1 STRAIN EDMONSTON 460MEASLES VIRUS NPDVSGPKL 1343 P10050.1 STRAIN HALLE 461 MEASLES VIRUSNPDVSGPKLTGALIG 1344 P04851.1 STRAIN EDMONSTON 462 MEASLES VIRUSNPDVSGPKLTGALIGILSLF 1345 P04851.1 STRAIN EDMONSTON 463 MEASLESNPPERIKLDYDQYCA 1346 P06830.1 MORBILLIVIRUS 464 MEASLES NQAKWAVPTTRTDDK1347 P06830.1 MORBILLIVIRUS 465 MEASLES NQDPDKILTYIAADH 1348 AAF02706.1MORBILLIVIRUS 466 MEASLES VIRUS NQLSCDLIGQKLGLK 1349 P69353.1 STRAINEDMONSTON 467 RUBELLA VIRUS NQQSRWGLGSPNCHGPDWASPVCQR 1350 ABD64214.1 HS468 MUMPS NSTLGVKSAREF 1351 ABP48111.1 RUBULAVIRUS 469 RUBELLA VIRUSNTPHGQLEVQVPPDP 1352 BAA28178.1 470 MEASLES VIRUS NVSAIFMAVLLTLQT 1353P69353.1 STRAIN EDMONSTON 471 MEASLES PAEVDGDVKLSSNLV 1354 P06830.1MORBILLIVIRUS 472 RUBELLA VIRUS PAFGHSDAACWGFPT 1355 BAA28178.1 473MEASLES VIRUS PALICCCRGRCNKKG 1356 P69353.1 STRAIN EDMONSTON 474 MEASLESPDKILTYIAADHC 1357 AAF02706.1 MORBILLIVIRUS 475 MEASLES VIRUSPERIKLDYDQYCADV 1358 P08362.1 STRAIN EDMONSTON 476 RUBELLA VIRUSPERPRLRL 1359 CAJ88851.1 477 RUBELLA VIRUS PGCATQAPVPVRLAG 1360BAA28178.1 478 RUBELLA VIRUS PGCATQAPVPVRLAGVRFESKIVDGG 1361 CAJ88851.1VACCINE STRAIN CFA RA27/3 479 RUBELLA VIRUS PGEVWVTP 1362 CAJ88851.1 480RUBELLA VIRUS PGEVWVTPVIGSQAR 1363 BAA28178.1 481 CORYNEBACTERIUMPGKLDVNKSKTHISVN 1364 CAE11230.1 DIPHTHERIAE 482 MEASLES VIRUSPGLGAPVFHMTNYLE 1365 P08362.1 STRAIN EDMONSTON 483 RUBELLA VIRUSPGPGEVWV 1366 CAJ88851.1 484 RUBELLA VIRUS PHKTVRVKFHTETRT 1367BAA28178.1 485 MEASLES PIELQVECFTWDQKL 1368 AAR89413.1 MORBILLIVIRUS 486MEASLES VIRUS PISLERLDVG 1369 P26031.1 STRAIN EDMONSTON-B 487MEASLES VIRUS PISLERLDVGTNLGN 1370 P69353.1 STRAIN EDMONSTON 488BORDETELLA PKALFTQQGGAYGRC 1371 P04979.1 PERTUSSIS 489 MEASLES VIRUSPKYVATQGYLISNFD 1372 P69353.1 STRAIN EDMONSTON 490 MEASLES VIRUSPLDIDTASESSQD 1373 P04851.1 STRAIN EDMONSTON 491 RUBELLA VIRUSPLGLKFKTVRPVALP 1374 BAA28178.1 492 MEASLES VIRUS PLITHGSGMDLYKSN 1375P08362.1 STRAIN EDMONSTON 493 MEASLES PLKDNRIPSYGVLSV 1376 P06830.1MORBILLIVIRUS 494 RUBELLA VIRUS PLLRTAPG 1377 CAJ88851.1 495MEASLES VIRUS PLLSCKPWQESRK 1378 P04851.1 STRAIN EDMONSTON 496BORDETELLA PPATVYRYDSRPPE 1379 P04977.1 PERTUSSIS 497 MEASLESPPISLERLDVGT 1380 AAL29688.1 MORBILLIVIRUS 498 RUBELLA VIRUSPPPPEERQETRSQTPAPKPS 1381 P07566.1 499 BORDETELLA PQEQITQHGSPYGRC 1382AAA83981.1 PERTUSSIS 500 BORDETELLA PQEQITQHGSPYGRCANK 1383 AAA83981.1PERTUSSIS 501 BORDETELLA PQPGPQPPQPPQPQPEAPAPQPPAG 1384 P14283.3PERTUSSIS 502 MEASLES VIRUS PRLGGKEDRRVKQ 1385 P04851.1 STRAIN EDMONSTON503 RUBELLA VIRUS PRLRLVDA 1386 CAJ88851.1 504 RUBELLA VIRUSPRNVRVTGCYQCGTP 1387 BAA28178.1 505 MEASLES VIRUS PSRASDARAAHLP 1388P04851.1 STRAIN EDMONSTON 506 MEASLES VIRUS PTGQIHWGNLSKIGV 1389P69353.1 STRAIN EDMONSTON 507 MEASLES VIRUS PTGTPLDIDTASE 1390 P04851.1STRAIN EDMONSTON 508 MEASLES VIRUS PTLSEIKGVIVHRLE 1391 P69353.1 STRAINEDMONSTON 509 MEASLES PTTIRGQFSNMSLSL 1392 P06830.1 MORBILLIVIRUS 510MEASLES PTTRTDDKLR 1393 AAR89413.1 MORBILLIVIRUS 511 MEASLESPTTRTDDKLRMETCFQQACKG 1394 AAR89413.1 MORBILLIVIRUS 512 RUBELLA VIRUSPVALPRTLAPPRNVR 1395 BAA28178.1 513 CORYNEBACTERIUM PVFAGANYAAWAVNVAQVI1396 AAV70486.1 DIPHTHERIAE 514 RUBELLA VIRUS PVIGSQAR 1397 CAJ88851.1515 MEASLES PVVEVNGVTIQVGSR 1398 AAL29688.1 MORBILLIVIRUS 516RUBELLA VIRUS PWELVVLTARPEDGWTCRGV 1399 P07566.1 STRAIN THERIEN 517MEASLES VIRUS PWQESRKNKAQTR 1400 P04851.1 STRAIN EDMONSTON 518MEASLES VIRUS PYMVNLENSIQNKFS 1401 P04851.1 STRAIN EDMONSTON 519MEASLES VIRUS PYMVNLENSIQNKFSAGSYP 1402 P04851.1 STRAIN EDMONSTON 520MEASLES VIRUS PYQGSGKGVS 1403 P08362.1 STRAIN EDMONSTON 521RUBELLA VIRUS PYQVSCGGESDRASA 1404 BAA28178.1 522 MEASLES QACKGKIQALCEN1405 P08362.1 MORBILLIVIRUS 523 MEASLES VIRUS QAGQEMILAVQGVQD 1406P69353.1 STRAIN EDMONSTON 524 MEASLES QALCENPECVPLKDN 1407 P06830.1MORBILLIVIRUS 525 BORDETELLA QALGALK 1408 ACI16088.1 PERTUSSIS 526RUBELLA VIRUS QAPVPVRLAGVRFES 1409 BAA28178.1 527 RUBELLA VIRUSQCGTPALVEGLAPGG 1410 BAA28178.1 528 MEASLES VIRUS QDPDKILTYIAADHC 1411P69353.1 STRAIN EDMONSTON 529 MEASLES VIRUS QECLRGSTKSCARTL 1412P69353.1 STRAIN EDMONSTON 530 BORDETELLA QEQITQHGSPYGRC 1413 AAA83981.1PERTUSSIS 531 MEASLES VIRUS QESRKNKAQTRTP 1414 P04851.1 STRAIN EDMONSTON532 MEASLES VIRUS QGDQSENELPRLGGK 1415 P04851.1 STRAIN EDMONSTON 533MEASLES VIRUS QGDQSENELPRLGGKEDRRV 1416 P04851.1 STRAIN EDMONSTON 534CORYNEBACTERIUM QGESGHDIKITAENTPLPIA 1417 AAV70486.1 DIPHTHERIAE 535MEASLES QGSGKGVSFQLVKLG 1418 P06830.1 MORBILLIVIRUS 536 MEASLES VIRUSQGVQDYINNELIPSM 1419 P69353.1 STRAIN EDMONSTON 537 RUBELLA VIRUSQLEVQVPPDPGDLVE 1420 BAA28178.1 538 MEASLES QLPEATFMV 1421 ABK40528.1MORBILLIVIRUS 539 RUBELLA VIRUS QLPFLGHDGHHGGTLRVGQHYRNAS 1422NP_740663.1 540 MEASLES VIRUS QLSMYRVFEV 1423 P08362.1 STRAIN EDMONSTON541 BORDETELLA QLSNIT 1424 ACI16083.1 PERTUSSIS 542 MEASLES VIRUSQNIRPVQSVASSRRH 1425 P69353.1 STRAIN EDMONSTON 543 MEASLES VIRUSQNKFSAGSYPLLWSY 1426 P04851.1 STRAIN EDMONSTON 544 MEASLES VIRUSQNKFSAGSYPLLWSYAMGVG 1427 P04851.1 STRAIN EDMONSTON 545 MEASLES VIRUSQNKFSAGSYPLLWSYAMGVGVELEN 1428 P04851.1 STRAIN EDMONSTON 546MEASLES VIRUS QQACKGKIQALCENP 1429 P08362.1 STRAIN EDMONSTON 547MEASLES VIRUS QQRRVVGEFRLERKW 1430 P04851.1 STRAIN EDMONSTON 548MEASLES VIRUS QQRRVVGEFRLERKWLDVVR 1431 P04851.1 STRAIN EDMONSTON 549BORDETELLA QQTRANPNPYTSRRSVAS 1432 P04977.1 PERTUSSIS 550 RUBELLA VIRUSQRHSPDCSRLVGATP 1433 BAA28178.1 551 MEASLES VIRUS QSGLTFASRGTNMED 1434P04851.1 STRAIN EDMONSTON 552 MEASLES VIRUS QSGLTFASRGTNMEDEADQY 1435P04851.1 STRAIN EDMONSTON 553 CORYNEBACTERIUM QSIALSSLMVAQAIPLVGEL 1436AAV70486.1 DIPHTHERIAE 554 MEASLES VIRUS QSRFGWFENKEISDI 1437 P04851.1STRAIN EDMONSTON 555 MEASLES VIRUS QSRFGWFENKEISDIEVQDP 1438 P04851.1STRAIN EDMONSTON 556 MEASLES VIRUS QSRGEAR 1439 P04851.1 STRAINEDMONSTON 557 MEASLES VIRUS QSRGEARESYRETGPSRA 1440 P04851.1 STRAINEDMONSTON 558 RUBELLA VIRUS QTGRGGSAPRPELGPPTN 1441 P07566.1STRAIN THERIEN 559 RUBELLA VIRUS QTPAPKPSRAPPQQPQPPRMQTGRG 1442 P07566.1STRAIN THERIEN 560 MEASLES VIRUS QVGSRRYPDAVYLHR 1443 P69353.1 STRAINEDMONSTON 561 MEASLES VIRUS QVSFLQGDQSENE 1444 P04851.1 STRAIN EDMONSTON562 RUBELLA VIRUS QYHPTACEVEPAFGH 1445 BAA28178.1 563 MEASLES VIRUSQYVLATYDTSRVEHA 1446 P08362.1 STRAIN EDMONSTON 564 BORDETELLARANPNPYTSRRSV 1447 ACI04548.1 PERTUSSIS 565 MEASLES VIRUS RASDARAAHLPTG1448 P04851.1 566 MEASLES VIRUS RASLETTNQAIEAIR 1449 P69353.1 STRAINEDMONSTON 567 RUBELLA VIRUS RCGRLICGLSTTAQYPPTRF 1450 P07566.1STRAIN THERIEN 568 BORDETELLA RDGQSVIGACASPYEGRYR 1451 P04979.1PERTUSSIS 569 MEASLES VIRUS RESYRETGPSRAS 1452 P04851.1 STRAIN EDMONSTON570 MEASLES VIRUS RETGPSRASDARA 1453 P04851.1 STRAIN EDMONSTON 571 MUMPSRFAKYQQQGRLEAR 1454 P21186.1 RUBULAVIRUS 572 RUBELLA VIRUSRFGAPQAFLAGLLLATVAVGTARA 1455 P07566.1 STRAIN THERIEN 573 MEASLES VIRUSRFMVALILDIKRTPG 1456 P04851.1 STRAIN EDMONSTON 574 MEASLES VIRUSRFMVALILDIKRTPGNKPRI 1457 P04851.1 STRAIN EDMONSTON 575 MEASLES VIRUSRGEARESYRETGP 1458 P04851.1 STRAIN EDMONSTON 576 RUBELLA VIRUS RGTTPPAYG1459 CAA28880.1 577 RUBELLA VIRUS RIETRSARH 1460 ABD64214.1 STRAIN M33578 BORDETELLA RILAGALATYQ 1461 P04977.1 PERTUSSIS 579 BORDETELLARIPPENIRRVT 1462 ACI04548.1 PERTUSSIS 580 BORDETELLA RISNLND 1463ACI16083.1 PERTUSSIS 581 BORDETELLA RLANLNG 1464 ACI16088.1 PERTUSSIS582 MEASLES VIRUS RLDVGTNLGNAIAKL 1465 P69353.1 STRAIN EDMONSTON 583MEASLES VIRUS RLERKWLDV 1466 P04851.1 STRAIN EDMONSTON 584 MEASLES VIRUSRLGGKEDRRVKQSRG 1467 P04851.1 STRAIN EDMONSTON 585 MEASLES VIRUSRLGGKEDRRVKQSRGEARES 1468 P04851.1 STRAIN EDMONSTON 586 MEASLES VIRUSRLLDRLVRL 1469 ABI54110.1 STRAIN EDMONSTON 587 RUBELLA VIRUS RLRLVDAD1470 CAJ88851.1 588 RUBELLA VIRUS RLRLVDADDPLLR 1471 BAA19893.1 589RUBELLA VIRUS RLRLVDADDPLLRTAPGPGEVWVTP 1472 BAA19893.1 VIGSQA 590RUBELLA VIRUS RLRLVQDADDPLLRIAPGPGEVWVTP 1473 SRC265968 VIGSQA 591MEASLES VIRUS RLSDNGYYTV 1474 ABK40528.1 STRAIN EDMONSTON 592RUBELLA VIRUS RLVDADDP 1475 CAJ88851.1 593 RUBELLA VIRUS RLVDADDPLLRTAPG1476 BAA28178.1 594 RUBELLA VIRUS RLVGATPE 1477 CAJ88851.1 595RUBELLA VIRUS RMQTGRGGSAPRPELGPPTNPFQAAV 1478 ABD64216.1 A 596 MEASLESRMSKGVFKV 1479 ABY21184.1 MORBILLIVIRUS 597 MEASLES RNPGLGAPVFHMTNY 1480P06830.1 MORBILLIVIRUS 598 RUBELLA VIRUS RPRLRLVD 1481 CAJ88851.1 599BORDETELLA RQAESSEAMAAWSERAGEA 1482 P04977.1 PERTUSSIS 600 RUBELLA VIRUSRQTWAEWAAAHWWQL 1483 BAA28178.1 601 MEASLES VIRUS RRVKQSRGEARES 1484P04851.1 STRAIN EDMONSTON 602 MEASLES RRYPDAVYL 1485 ACA09725.1MORBILLIVIRUS 603 MEASLES VIRUS RRYPDAVYLHRIDLG 1486 P69353.1 STRAINEDMONSTON 604 MEASLES VIRUS RSAGKVSSTLASELG 1487 P04851.1 STRAINEDMONSTON 605 MEASLES VIRUS RSAGKVSSTLASELGITAED 1488 P04851.1 STRAINEDMONSTON 606 MEASLES VIRUS RSELSQLS 1489 P08362.1 STRAIN EDMONSTON 607MEASLES VIRUS RSELSQLSMYRVFEV 1490 P08362.1 STRAIN EDMONSTON 608RUBELLA VIRUS RSQTPAPKPSRAPPQQPQPPRMQT 1491 ABD64214.1 609 RUBELLA VIRUSRTAPGPGE 1492 CAJ88851.1 610 RUBELLA VIRUS RTAPGPGEVWVTPVI 1493BAA28178.1 611 MEASLES RTDDKLRMETCFQQA 1494 P06830.1 MORBILLIVIRUS 612RUBELLA VIRUS RTLAPPRNVRVTGCY 1495 BAA28178.1 613 MEASLES VIRUSRTVLEPIRDALNAMT 1496 P69353.1 STRAIN EDMONSTON 614 MEASLES VIRUSRVEHAVVYYVYSPSR 1497 P08362.1 STRAIN EDMONSTON 615 MEASLES VIRUSRVFEVGVIRNPGLGA 1498 P08362.1 STRAIN EDMONSTON 616 BORDETELLARVHVSKEEQYYDYEDATFE 1499 P04978.2 PERTUSSIS 617 RUBELLA VIRUS RVIDPAAQ1500 BAA28178.1 618 RUBELLA VIRUS RVKFHTETRTVWQLS 1501 BAA28178.1 619BORDETELLA RVYHNGITGET 1502 ACI04548.1 PERTUSSIS 620 BORDETELLARYDSRPPEDVF 1503 ACI04548.1 PERTUSSIS 621 MEASLES VIRUS RYPDAVYLHRIDLGP1504 P69353.1 STRAIN EDMONSTON 622 MEASLES VIRUS SAEISIQALSYALGG 1505P69353.1 STRAIN EDMONSTON 623 MEASLES VIRUS SAEPLLSCKPWQESR 1506P04851.1 STRAIN EDMONSTON 624 MEASLES VIRUS SAEPLLSCKPWQESRKNKAQ 1507P04851.1 STRAIN EDMONSTON 625 MEASLES SAGKVSSTLASELG 1508 P04851.1MORBILLIVIRUS 626 MEASLES VIRUS SAGKVSSTLASELGITAEDARLVS 1509 P04851.1STRAIN EDMONSTON 627 MEASLES VIRUS SCTVTREDGT 1510 P08362.1 STRAINEDMONSTON 628 RUBELLA VIRUS SDAACWGFPTDTVMS 1511 BAA28178.1 629MEASLES VIRUS SDARAAHLPTGTP 1512 P04851.1 STRAIN EDMONSTON 630RUBELLA VIRUS SDWHQGTHVCHTKHMDFWCVEHD 1513 P07566.1 STRAIN THERIEN 631MEASLES VIRUS SELRRWIKYTQQRRV 1514 P04851.1 STRAIN EDMONSTON 632MEASLES VIRUS SELRRWIKYTQQRRVVGEFR 1515 P04851.1 STRAIN EDMONSTON 633MEASLES VIRUS SELSQL 1516 P08362.1 STRAIN EDMONSTON 634 MEASLES VIRUSSELSQLS 1517 P08362.1 STRAIN EDMONSTON 635 BORDETELLASEYLAHRRIPPENIRRVTRV 1518 CAD44970.1 PERTUSSIS 636 MEASLES VIRUSSFLQGDQSENELP 1519 P04851.1 STRAIN EDMONSTON 637 MEASLES VIRUSSGKGVSFQLVKLGVW 1520 P08362.1 STRAIN EDMONSTON 638 MEASLES VIRUSSHRGVIADNQAKWAV 1521 P08362.1 STRAIN EDMONSTON 639 MEASLES VIRUSSIEHQVKDVLTPLFK 1522 P08362.1 STRAIN EDMONSTON 640 MEASLES VIRUSSKIGVVGIGSASYKV 1523 P69353.1 STRAIN EDMONSTON 641 MEASLESSKRSELSQLSMYRVF 1524 P06830.1 MORBILLIVIRUS 642 MEASLESSLFVESPGQLIQRITDDPDVS 1525 ABI54110.1 MORBILLIVIRUS 643 MEASLES VIRUSSLSTNLDVTNSIEHQ 1526 P08362.1 STRAIN EDMONSTON 644 MEASLES VIRUSSLSTNLDVTNSIEHQVKDVLTPLFK 1527 P08362.1 STRAIN EDMONSTON 645MEASLES VIRUS SLWGSGLLML 1528 BAE98296.1 STRAIN EDMONSTON 646MEASLES VIRUS SMKGLSSTSIVYILI 1529 P69353.1 STRAIN EDMONSTON 647 MEASLESSMYRVFEVGV 1530 P08362.1 MORBILLIVIRUS 648 MEASLES SNDLSNCMVALGELK 1531P06830.1 MORBILLIVIRUS 649 MEASLES VIRUS SPGQLIQR 1532 P10050.1STRAIN HALLE 650 MEASLES VIRUS SQDPQDSRRSAEP 1533 P04851.1 STRAINEDMONSTON 651 MEASLES SRIVINREHLMIDRP 1534 P06830.1 MORBILLIVIRUS 652MEASLES VIRUS SRKNKAQTRTPLQ 1535 P04851.1 STRAIN EDMONSTON 653RUBELLA VIRUS SRLVGATP 1536 CAJ88851.1 654 RUBELLA VIRUSSRLVGATPERPRLRLVDADDPLLR 1537 CAJ88851.1 655 MEASLES VIRUSSRPGLKPDLTGTSKS 1538 P69353.1 STRAIN EDMONSTON 656 BORDETELLASRRSVASIVGTLVRM 1539 CAD44970.1 PERTUSSIS 657 RUBELLA VIRUSSRWGLGSPNCHGPDW 1540 BAA28178.1 658 BORDETELLA SSATK 1541 ACI16088.1PERTUSSIS 659 RUBELLA VIRUS SSGGYAQLASYFNPG 1542 BAA28178.1 660BORDETELLA SSLGNGV 1543 ACI16083.1 PERTUSSIS 661 MEASLES SSNLVILPGQDLQYV1544 P06830.1 MORBILLIVIRUS 662 MEASLES VIRUS SSQDPQDSRRSAEPL 1545P04851.1 STRAIN EDMONSTON 663 MEASLES VIRUS SSQDPQDSRRSAEPLLSCKP 1546P04851.1 STRAIN EDMONSTON 664 MEASLES VIRUS SSRRHKRFAGVVLAG 1547P69353.1 STRAIN EDMONSTON 665 MEASLES VIRUS SSTSIVYILIAVCLG 1548P69353.1 STRAIN EDMONSTON 666 BORDETELLA STPGIVI 1549 AAA83981.1PERTUSSIS 667 BORDETELLA STPGIVIPPQEQITQHGSPYGRC 1550 AAA83981.1PERTUSSIS 668 BORDETELLA STSSSRRYTEVY 1551 P04977.1 PERTUSSIS 669MEASLES VIRUS SYFIVLSIAYPTLSE 1552 P69353.1 STRAIN EDMONSTON 670MEASLES VIRUS SYRETGPSRASDA 1553 P04851.1 STRAIN EDMONSTON 671BORDETELLA SYVK 1554 ACI16083.1 PERTUSSIS 672 RUBELLA VIRUSSYVQHPHKTVRVKFH 1555 BAA28178.1 673 RUBELLA VIRUS TAPGPGEV 1556CAJ88851.1 674 BORDETELLA TATRLLSSTNSRLC 1557 AAA83981.1 PERTUSSIS 675MEASLES TDDPVIDRLYLSSHR 1558 P06830.1 MORBILLIVIRUS 676 MEASLES VIRUSTEILSLFGPSLRDPI 1559 P69353.1 STRAIN EDMONSTON 677 RUBELLA VIRUSTETRTVWQLSVAGVS 1560 BAA28178.1 678 BORDETELLA TEVYLEHRMQEAVE 1561P04977.1 PERTUSSIS 679 MEASLES VIRUS TFMPEGTVCSQNALY 1562 P69353.1STRAIN EDMONSTON 680 RUBELLA VIRUS TGACICEIPTDVSCE 1563 BAA28178.1 681BORDETELLA TGDLRAY 1564 ACI16083.1 PERTUSSIS 682 MEASLES TGMQSWVPLSTDDPV1565 P06830.1 MORBILLIVIRUS 683 RUBELLA VIRUS TGNQQSRWGLGSPNC 1566BAA28178.1 684 MEASLES VIRUS TGPSRASDARAAH 1567 P04851.1 STRAINEDMONSTON 685 MEASLES TGTIINQDPDKILTY 1568 AAF02706.1 MORBILLIVIRUS 686RUBELLA VIRUS TGVVYGTHTTAVSET 1569 BAA28178.1 687 MEASLES VIRUSTIRGQFSNMSLSLLD 1570 P08362.1 STRAIN EDMONSTON 688 RUBELLA VIRUSTLGATCALPLAGLLA 1571 BAA28178.1 689 MEASLES TLLNNCTRV 1572 P26031.1MORBILLIVIRUS 690 MEASLES VIRUS TLNVPPPPDPGR 1573 P03422.1 STRAINEDMONSTON-B 691 MEASLES VIRUS TLNVPPPPDPGRASTSGTPIKK 1574 P03422.1STRAIN EDMONSTON-B 692 MEASLES TMTSQGMYGGTYPVE 1575 P06830.1MORBILLIVIRUS 693 MEASLES VIRUS TNLGNAIAKLEDAKE 1576 P69353.1 STRAINEDMONSTON 694 MEASLES VIRUS TNMEDEADQYFSHDD 1577 P04851.1 STRAINEDMONSTON 695 MEASLES VIRUS TNMEDEADQYFSHDDPISSD 1578 P04851.1 STRAINEDMONSTON 696 RUBELLA VIRUS TNPFQAAVARGLRPP 1579 CAA28880.1 STRAIN M33697 MEASLES TNSIEHQVKDVLTPL 1580 P06830.1 MORBILLIVIRUS 698MEASLES VIRUS TNYLEQPVSNDLSNC 1581 P08362.1 STRAIN EDMONSTON 699 MEASLESTNYLEQPVSNDLSNCMVALGELKLA 1582 AAR89413.1 MORBILLIVIRUS AL 700RUBELLA VIRUS TPERPRLR 1583 CAJ88851.1 701 RUBELLA VIRUSTPERPRLRLVDADDPLLRTA 1584 P07566.1 STRAIN THERIEN 702 MEASLES VIRUSTPGNKPRIA 1585 P10050.1 STRAIN HALLE 703 MEASLES VIRUS TPLDIDTASESSQDP1586 P04851.1 STRAIN EDMONSTON 704 MEASLES VIRUS TPLDIDTASESSQDPQDSRR1587 P04851.1 STRAIN EDMONSTON 705 MEASLES VIRUS TPLFKIIGDEVGLRT 1588P08362.1 STRAIN EDMONSTON 706 MEASLES VIRUS TPLQCTM 1589 P04851.1 STRAINEDMONSTON 707 RUBELLA VIRUS TPVIGSQA 1590 CAJ88851.1 708 RUBELLA VIRUSTPVIGSQARK 1591 BAA19893.1 709 MEASLES VIRUS TQGYLISNFDESSCT 1592P69353.1 STRAIN EDMONSTON 710 BORDETELLA TRANPNPYTSRRSVASIVGTLVRM 1593P04977.1 PERTUSSIS 711 RUBELLA VIRUS TRFGCAMRWGLPP 1594 NP_740663.1 712BORDETELLA TRNTGQPATDHYYSNV 1595 AAA83981.1 PERTUSSIS 713 MEASLES VIRUSTRTPLQCTMTEIF 1596 P04851.1 STRAIN EDMONSTON 714 RUBELLA VIRUSTRWHRLLRMPVR 1597 ABD64216.1 715 MEASLES VIRUS TSGSGGAIRGIKHII 1598P04851.1 STRAIN EDMONSTON 716 MEASLES VIRUS TSGSGGAIRGIKHIIIVPIP 1599P04851.1 STRAIN EDMONSTON 717 MEASLES VIRUS TSQGMYGGTYLVEKP 1600P08362.1 STRAIN EDMONSTON 718 MEASLES TSRVEHAVVYYVYSP 1601 P06830.1MORBILLIVIRUS 719 BORDETELLA TSSSRRYTEVYL 1602 ACI04548.1 PERTUSSIS 720BORDETELLA TSYVG 1603 ACI16088.1 PERTUSSIS 721 MEASLES VIRUSTTEDKISRAVGPRQA 1604 P04851.1 STRAIN EDMONSTON 722 MEASLES VIRUSTTEDKISRAVGPRQAQVSFL 1605 P04851.1 STRAIN EDMONSTON 723 RUBELLA VIRUSTTERIETRSARHP 1606 ABD64214.1 STRAIN M33 724 MEASLES VIRUSTTNQAIEAIRQAGQE 1607 P69353.1 STRAIN EDMONSTON 725 RUBELLA VIRUSTTSDPWHPPGPLGLK 1608 BAA28178.1 726 MEASLES VIRUS TVCSQNALYPMSPLL 1609P69353.1 STRAIN EDMONSTON 727 RUBELLA VIRUS TVNGEDVGAVPPGKF 1610BAA28178.1 728 MEASLES TYPVEKPNLSSKRSE 1611 P06830.1 MORBILLIVIRUS 729RUBELLA VIRUS VAGVSCNVTTEHPFC 1612 BAA28178.1 730 BORDETELLAVAPGIVIPPKALFTQQGGAYGRC 1613 P04979.1 PERTUSSIS 731 RUBELLA VIRUSVCHTKHMDFWCVEHDRPPPATPTPL 1614 NP_740663.1 732 RUBELLA VIRUSVCQRHSPDCSRLVGATPER 1615 BAA19893.1 733 RUBELLA VIRUS VDADDPLL 1616CAJ88851.1 734 RUBELLA VIRUS VDADDPLLRTAPGPGEVWVT 1617 BAA19893.1 735CORYNEBACTERIUM VDIGFAAYNFVESIINLFQV 1618 AAV70486.1 DIPHTHERIAE 736MEASLES VIRUS VEIAEYRRLLRTVLE 1619 P69353.1 STRAIN EDMONSTON 737MEASLES VIRUS VELENSMGGLNFGRS 1620 P04851.1 STRAIN EDMONSTON 738MEASLES VIRUS VELENSMGGLNFGRSYFDPA 1621 P04851.1 STRAIN EDMONSTON 739MEASLES VELKIKIASGFGPLI 1622 P06830.1 MORBILLIVIRUS 740 RUBELLA VIRUSVEMDEWIHAHTTSD 1623 SRC265968 741 RUBELLA VIRUS VEMPEWIHAHTTSDP 1624BAA28178.1 742 CORYNEBACTERIUM VERRLVKVL 1625 P33120.2 DIPHTHERIAE 743MEASLES VESPGQLI 1626 ABI54110.1 MORBILLIVIRUS 744 MEASLES VIRUSVESPGQLIQRITDDP 1627 P04851.1 STRAIN EDMONSTON 745 MEASLES VIRUSVESPGQLIQRITDDPDVSIR 1628 P04851.1 STRAIN EDMONSTON 746 RUBELLA VIRUSVFALASYVQHPHKTV 1629 BAA28178.1 747 RUBELLA VIRUS VGATPERP 1630CAJ88851.1 748 RUBELLA VIRUS VGATPERPRL 1631 BAA19893.1 749RUBELLA VIRUS VGATPERPRLRLVDA 1632 BAA28178.1 750 MEASLES VIRUSVGIGSASYKVMTRSS 1633 P69353.1 STRAIN EDMONSTON 751 MEASLES VIRUSVGLRTPQRFTDLVKF 1634 P08362.1 STRAIN EDMONSTON 752 CORYNEBACTERIUMVHHNTEEIVAQSIALSSLMV 1635 AAV70486.1 DIPHTHERIAE 753 MEASLES VIRUSVHRLEGVSYNIGSQE 1636 P69353.1 STRAIN EDMONSTON 754 RUBELLA VIRUSVIGSQARK 1637 CAJ88851.1 755 BORDETELLA VITGSI 1638 ACI16088.1 PERTUSSIS756 BORDETELLA VITGTI 1639 ACI16083.1 PERTUSSIS 757 MEASLES VIRUSVKQSRGEA 1640 P04851.1 STRAIN EDMONSTON 758 MEASLES VIRUS VLFVMFLSLI1641 P08362.1 STRAIN EDMONSTON 759 MEASLES VLFVMFLSLIGLLAI 1642 P06830.1MORBILLIVIRUS 760 MEASLES VLTPLFKIIGDEVGL 1643 P06830.1 MORBILLIVIRUS761 MEASLES VIRUS VMTRSSHQSLVIKLM 1644 P69353.1 STRAIN EDMONSTON 762RUBELLA VIRUS VPAAPCARIWNGTQR 1645 BAA28178.1 763 RUBELLA VIRUSVPPDPGDLVEYIMNY 1646 BAA28178.1 764 MEASLES VIRUS VQSVASSRRHKRFAG 1647P69353.1 STRAIN EDMONSTON 765 BORDETELLA VQTGGTSRTVTMRYLAS 1648ACI16083.1 PERTUSSIS 766 BORDETELLA VQVRI 1649 ACI16083.1 PERTUSSIS 767RUBELLA VIRUS VRAYNQPAGDV 1650 NP_740662.1 768 RUBELLA VIRUSVRFESKIVDGGCFAP 1651 BAA28178.1 769 RUBELLA VIRUS VRLAGVRFESKIVDG 1652BAA28178.1 770 MEASLES VIRUS VSGSFGNRFILSQGN 1653 P69353.1 STRAINEDMONSTON 771 BORDETELLA VSKEEQYYDYEDAT 1654 AAA83981.1 PERTUSSIS 772MEASLES VIRUS VSKGNCSGPTTIRGQ 1655 P08362.1 STRAIN EDMONSTON 773RUBELLA VIRUS VTAALLNTPPPYQVS 1656 BAA28178.1 774 RUBELLA VIRUSTGCYQCGTPALVEG 1657 BAA28178.1 775 RUBELLA VIRUS VTPVIGSQ 1658CAJ88851.1 776 RUBELLA VIRUS VTTEHPFCNTPHGQLEVQVPPD 1659 P07566.1STRAIN THERIEN 777 MEASLES VIRUS VVLAGAALGVATAAQ 1660 P69353.1 STRAINEDMONSTON 778 RUBELLA VIRUS VWQLSVAGVSCNVTT 1661 BAA28178.1 779RUBELLA VIRUS VWVTPVIG 1662 CAJ88851.1 780 RUBELLA VIRUS VWVTPVIGSQAR1663 BAA19893.1 781 MEASLES VIRUS VYILIAVCLGGLIGI 1664 P69353.1 STRAINEDMONSTON 782 MEASLES VIRUS VYLHRIDLGPPISLE 1665 P69353.1 STRAINEDMONSTON 783 BORDETELLA VYRYDSRP 1666 P04977.1 PERTUSSIS 784 BORDETELLAVYRYDSRPPEDV 1667 P04977.1 PERTUSSIS 785 MEASLES VYWLTIPPMKNLALG 1668P06830.1 MORBILLIVIRUS 786 RUBELLA VIRUS WDLEATGACICEIPT 1669 BAA28178.1787 MEASLES WDQKLWCRHFCVLAD 1670 AAR89413.1 MORBILLIVIRUS 788RUBELLA VIRUS WGFPTDTVMSVFALA 1671 BAA28178.1 789 RUBELLA VIRUSWHPPGPLGLKFKTVR 1672 BAA28178.1 790 RUBELLA VIRUS WIHAHTTSDPWHPPG 1673BAA28178.1 791 MEASLES VIRUS WLTIPPMKNLALGVI 1674 P08362.1 STRAINEDMONSTON 792 MEASLES VIRUS WQESRKNKAQTRTPLQCTMT 1675 P04851.1 STRAINEDMONSTON 793 RUBELLA VIRUS WVCIFMVCRRACR 1676 SRC265968 794RUBELLA VIRUS WVTPVIGS 1677 CAJ88851.1 795 MEASLES VIRUS WYTTVPKYVATQGYL1678 P69353.1 STRAIN EDMONSTON 796 MEASLES VIRUS YALGGDINKVLEKLG 1679P69353.1 STRAIN EDMONSTON 797 MEASLES VIRUS YAMGVGVELE 1680 P04851.1STRAIN EDMONSTON 798 MEASLES YAMGVGVELEN 1681 ABI54110.1 MORBILLIVIRUS799 MEASLES YCADVAAEELMNALV 1682 AAR89413.1 MORBILLIVIRUS 800 MEASLESYDFRDLTWCINPPER 1683 P06830.1 MORBILLIVIRUS 801 BORDETELLA YFEPGPT 1684ACI16083.1 PERTUSSIS 802 RUBELLA VIRUS YFNPGGSYYKQYHPT 1685 BAA28178.1803 MEASLES VIRUS YFRLGQEMVRRSAGK 1686 P04851.1 STRAIN EDMONSTON 804MEASLES VIRUS YFRLGQEMVRRSAGKVSSTL 1687 P04851.1 STRAIN EDMONSTON 805MEASLES YFYPFRLPIKGVPIE 1688 P06830.1 MORBILLIVIRUS 806 BORDETELLAYGDNAGRILAGALAT 1689 P04977.1 PERTUSSIS 807 RUBELLA VIRUSYGHATVEMPEWIHAH 1690 BAA28178.1 808 RUBELLA VIRUS YIMNYTGNQQSRWGL 1691BAA28178.1 809 MEASLES VIRUS YINNELIPSMNQLSC 1692 P69353.1 STRAINEDMONSTON 810 RUBELLA VIRUS YLCTAPGCATQAPVP 1693 BAA28178.1 811 MEASLESYLFTVPIKEAGEDCH 1694 P06830.1 MORBILLIVIRUS 812 MEASLES VIRUS YLHDPEFNL1695 ABK40531.1 STRAIN EDMONSTON 813 MEASLES VIRUS YLNMSRLFV 1696ABK40531.1 STRAIN EDMONSTON 814 MEASLES VIRUS YPALGLHEF 1697 P10050.1STRAIN HALLE 815 MEASLES YPALGLHEFA 1698 ABI54110.1 MORBILLIVIRUS 816MEASLES VIRUS YPALGLHEFAGELST 1699 P04851.1 STRAIN EDMONSTON 817MEASLES VIRUS YPALGLHEFAGELSTLESLM 1700 P04851.1 STRAIN EDMONSTON 818MEASLES VIRUS YPFRLPIKGVPIELQ 1701 P08362.1 STRAIN EDMONSTON 819MEASLES VIRUS YPLLWSYAM 1702 P10050.1 STRAIN HALLE 820 MUMPS YQQQGRL1703 P21186.1 RUBULAVIRUS 821 BORDETELLA YQSEYLAHRR 1704 P04977.1PERTUSSIS 822 MEASLES VIRUS YRETGPSRASDARAA 1705 P04851.1 STRAINEDMONSTON 823 MEASLES VIRUS YRETGPSRASDARAAHLPTG 1706 P04851.1 STRAINEDMONSTON 824 RUBELLA VIRUS YRNASDVLPGHWLQGGWGCYNLSDW 1707 NP_740663.1825 MEASLES VIRUS YRRLLRTVLEPIRDA 1708 P69353.1 STRAIN EDMONSTON 826BORDETELLA YRYDSRPP 1709 P04977.1 PERTUSSIS 827 MEASLES VIRUSYSGGDLLGILESRGI 1710 P69353.1 STRAIN EDMONSTON 828 BORDETELLAYSKVTATBLLASTNSRLCAVFVRDG 1711 SRC280066 PERTUSSIS 829 MEASLES VIRUSYSPSRSFSYFYPFRL 1712 P08362.1 STRAIN EDMONSTON 830 RUBELLA VIRUSYTGNQQSRWGLGSPNCHGPDWASPV 1713 P07566.1 STRAIN THERIEN 831 MEASLESYVLLAVLFV 1714 P08362.1 MORBILLIVIRUS 832 MEASLES YVYSPGRSFSYFYPF 1715P06830.1 MORBILLIVIRUS 833 BORDETELLA YYDYEDATFQTYALTGISLCNPAASIC 1716P04979.1 PERTUSSIS 834 MEASLES GDLLGILESRGIKAR 1717 AAF02706.1MORBILLIVIRUS 835 MEASLES TVPKYVATQGYLISN 1718 AAL29688.1 MORBILLIVIRUS836 MEASLES KPWDSPQEI 1719 P26035.1 MORBILLIVIRUS 837 MEASLES KPWESPQEI1720 CAA34579.1 MORBILLIVIRUS 838 BORDETELLA ATYQSEYLAHRRIPP 1721ACI04548.1 PERTUSSIS 839 BORDETELLA CMARQAESSEAMAAWSERAGEAMV 1722ACI04548.1 PERTUSSIS LVYYESIAYSF 840 BORDETELLA CQVGSSNSAFVSTSSSRRYTEVYL1723 ACI04548.1 PERTUSSIS 841 BORDETELLA DDPPATVYRYDSRPP 1724 ACI04548.1PERTUSSIS 842 BORDETELLA GALATYQSEYLAHRRIPP 1725 ACI04548.1 PERTUSSIS843 BORDETELLA MAAWSERAGEAMVLVYYESIAYSF 1726 ACI04548.1 PERTUSSIS 844BORDETELLA MVLVYYESIAYSF 1727 ACI04548.1 PERTUSSIS 845 BORDETELLAPATVYRYDSRPPEDV 1728 ACI04548.1 PERTUSSIS 846 BORDETELLA YDSRPPEDV 1729ACI04548.1 PERTUSSIS 847 BORDETELLA EPGITTNYDT 1730 ACI16087.1 PERTUSSIS848 BORDETELLA GDLRAYKMVYATNPQTQLSN 1731 ACI16083.1 PERTUSSIS 849BORDETELLA KNGDVEASAITTYVGFSVVYP 1732 ACI16083.1 PERTUSSIS 850BORDETELLA KVTNGSKSYTLRYLASYVK 1733 ACI16088.1 PERTUSSIS 851 BORDETELLAQALGALKLYFEPGITTNYDTGDLIAY 1734 ACI16088.1 PERTUSSIS KQTYNASGN 852BORDETELLA YATNPQTQLS 1735 ACI16083.1 PERTUSSIS 853 CORYNEBACTERIUMDNENPLSGKAGGVVKVTYPGLTKV 1736 AAV70486.1 DIPHTHERIAE 854 CORYNEBACTERIUMENFSSYHGTKPGYVDSI 1737 AAV70486.1 DIPHTHERIAE 855 CORYNEBACTERIUMKVDNAETIKKELGLSLTEP 1738 AAV70486.1 DIPHTHERIAE 856 RUBELLA VIRUSMEDLQKALEAQSRALRAGLAA 1739 CAA28880.1 STRAIN M33 857 CORYNEBACTERIUMQKGIQKPKSGTQGNYDDDWKGFY 1740 AAV70486.1 DIPHTHERIAE 858 RUBELLA VIRUSRTGAWQRKDWSRAPPPPEERQESRS 1741 CAA28880.1 STRAIN M33 QTPAPKPSR 859RUBELLA VIRUS AAGASQSRRPRPPRHARAQHLPEMT 1742 SRC265968 PAVT 860RUBELLA VIRUS CVTSWLWSEGEGAVFYRVDLHFINL 1743 CAA28880.1 GTP 861RUBELLA VIRUS FRVGGTRWHRLLRMPVRGLDGDTAP 1744 CAA28880.1 LP 862CORYNEBACTERIUM GRKIRMRCRAIDGDVTFCRPKSPVYV 1745 1007216A DIPHTHERIAE GN863 RUBELLA VIRUS GTPPLDEDGRWDPALMYNPCGPEPPA 1746 CAA28880.1 HV 864CORYNEBACTERIUM GVHANLHVAFHRSSSEKIHSNEISSDS 1747 AAV70486.1 DIPHTHERIAEIGVLGYQKTVDHTKVNSKLSLFFEIK S 865 RUBELLA VIRUS MASTTPITMEDLQKALEAQSRALRA1748 ABD64200.1 GLAAG 866 RUBELLA VIRUS PELGPPTNPFQAAVARGLRPPLHDPD 1749CAA28880.1 TEAPTEAC 867 RUBELLA VIRUS PLPPHTTERIETRSARHPWRIRFGAP 1750CAA28880.1 868 RUBELLA VIRUS SRAPPPPEERQESRSQTPAPKPSRAPP 1751 CAA28880.1869 RUBELLA VIRUS SRAPPQQPQPPRMQTGRGGSAPRPEL 1752 CAA28880.1 GP 870RUBELLA VIRUS TPAVTPEGPAPPRTGAWQRKDWSRAP 1753 CAA28880.1 P 871RUBELLA VIRUS VRAYNQPAGDVRGVWGKGERTYAE 1754 CAA28880.1 QDFRV 872RUBELLA VIRUS AFGHSDAACWGFPTDTVMSV 1755 CAA28880.1 873 RUBELLA VIRUSCARIWNGTQRACTFWAVNAYS 1756 CAA28880.1 874 RUBELLA VIRUSEEAFTYLCTAPGCATQTPVPVR 1757 CAA28880.1 875 RUBELLA VIRUSFAPWDLEATGACICEIPTDV 1758 CAA28880.1 876 RUBELLA VIRUS GEDVGAFPPGKFVTAAL1759 CAA28880.1 877 RUBELLA VIRUS GEVWVTPVIGSQARKCGLHI 1760 CAA28880.1878 RUBELLA VIRUS GQLEVQVPPDPGDLVEYIMN 1761 CAA28880.1 879 RUBELLA VIRUSGSYYKQYHPTACEVEPAFGH 1762 CAA28880.1 880 RUBELLA VIRUSIHAHTTSDPWHPPGPLGLKF 1763 CAA28880.1 881 RUBELLA VIRUSIMNYTGNQQSRWGLGSPNCH 1764 CAA28880.1 882 RUBELLA VIRUSLHIRAGPYGHATVEMPEWIH 1765 CAA28880.1 883 RUBELLA VIRUSLKFKTVRPVALPRALAPPRN 1766 CAA28880.1 884 RUBELLA VIRUSLNTPPPYQVSCGGESDRASAGH 1767 CAA28880.1 885 RUBELLA VIRUSNCHGPDWASPVCQRHSPDCS 1768 CAA28880.1 886 RUBELLA VIRUSPDCSRLVGATPERPRLRLVD 1769 CAA28880.1 887 RUBELLA VIRUSPRNVRVTGCYQCGTPALVEG 1770 CAA28880.1 888 RUBELLA VIRUSPTDVSCEGLGAWVPTAPCARI 1771 CAA28880.1 889 RUBELLA VIRUSRLVDADDPLLRTAPGPGEVW 1772 CAA28880.1 890 RUBELLA VIRUSSVFALASYVQHPHKTVRVKF 1773 CAA28880.1 891 RUBELLA VIRUSVEGLAPGGGNCHLTVNGEDV 1774 CAA28880.1 892 RUBELLA VIRUSVKFHTETRTVWQLSVAGVSC 1775 CAA28880.1 893 RUBELLA VIRUSVPVRLAGVGFESKIVDGGCF 1776 CAA28880.1 894 RUBELLA VIRUSVSCNVTTEHPFCNTPHGQLE 1777 CAA28880.1 895 BORDETELLA AAASSPDAHVPF 1778AAA22983.1 PERTUSSIS 896 BORDETELLA AASSPDA 1779 AAA22983.1 PERTUSSIS897 BORDETELLA AKLGAAASSPDA 1780 AAA22983.1 PERTUSSIS 898 BORDETELLAAMKPYEVTPTRM 1781 AAA22983.1 PERTUSSIS 899 BORDETELLAAMTHLSPALADVPYVLVKTNMVVTS 1782 AAA22983.1 PERTUSSIS 900 BORDETELLAASSPDAHVPFCF 1783 AAA22983.1 PERTUSSIS 901 BORDETELLAASSPDAHVPFCFGKDLKRPGSSPME 1784 AAA22983.1 PERTUSSIS 902 BORDETELLACFGKDLKRPGSS 1785 AAA22983.1 PERTUSSIS 903 BORDETELLACFGKDLKRPGSSPMEVMLRAVFMQQ 1786 AAA22983.1 PERTUSSIS 904 BORDETELLACGIAAKLGAAAS 1787 AAA22983.1 PERTUSSIS 905 BORDETELLACGIAAKLGAAASSPDAHVPFCFGKD 1788 AAA22983.1 PERTUSSIS 906 BORDETELLADAHVPFCFGKDL 1789 AAA22983.1 PERTUSSIS 907 BORDETELLA DLKRPGSSPMEV 1790AAA22983.1 PERTUSSIS 908 BORDETELLA DVPYVLVKTNMV 1791 AAA22983.1PERTUSSIS 909 BORDETELLA DVPYVLVKTNMVVTSVAMKPYEVTP 1792 AAA22983.1PERTUSSIS T 910 BORDETELLA EVMLRAVFMQQR 1793 AAA22983.1 PERTUSSIS 911BORDETELLA FEGKPALELIRM 1794 AAA22983.1 PERTUSSIS 912 BORDETELLAFLGPKQLTFEGK 1795 AAA22983.1 PERTUSSIS 913 BORDETELLAFLGPKQLTFEGKPALELIRMVECSG 1796 AAA22983.1 PERTUSSIS 914 BORDETELLAFMQQRPLRMFLGPKQLT 1797 AAA22983.1 PERTUSSIS 915 BORDETELLA GKDLKRPGSSPM1798 AAA22983.1 PERTUSSIS 916 BORDETELLA GKDLKRPGSSPME 1799 AAA22983.1PERTUSSIS 917 BORDETELLA GKPALELIRMVE 1800 AAA22983.1 PERTUSSIS 918BORDETELLA GPKQLTFEGKPA 1801 AAA22983.1 PERTUSSIS 919 BORDETELLAHVPFCFGKDLKR 1802 AAA22983.1 PERTUSSIS 920 BORDETELLA IAAKLGAAASSP 1803AAA22983.1 PERTUSSIS 921 BORDETELLA KPYEVTPTRMLV 1804 AAA22983.1PERTUSSIS 922 BORDETELLA KQLTFEGKPALE 1805 AAA22983.1 PERTUSSIS 923BORDETELLA KRPGSSPMEVML 1806 AAA22983.1 PERTUSSIS 924 BORDETELLALELIRMVECSGK 1807 AAA22983.1 PERTUSSIS 925 BORDETELLA LGAAASSPDAHV 1808AAA22983.1 PERTUSSIS 926 BORDETELLA LIRMVECSGKQD 1809 AAA22983.1PERTUSSIS 927 BORDETELLA LVCGIAAKLGAA 1810 AAA22983.1 PERTUSSIS 928BORDETELLA MKPYEVTPTRM 1811 AAA22983.1 PERTUSSIS 929 BORDETELLAMLRAVFMQQRPL 1812 AAA22983.1 PERTUSSIS 930 BORDETELLA MQQRPLRM 1813AAA22983.1 PERTUSSIS 931 BORDETELLA MQQRPLRMFLGP 1814 AAA22983.1PERTUSSIS 932 BORDETELLA MVVTSVAMKPYE 1815 AAA22983.1 PERTUSSIS 933BORDETELLA MVVTSVAMKPYEVTPTRMLVCGIAA 1816 AAA22983.1 PERTUSSIS 934BORDETELLA PALELIRMVECS 1817 AAA22983.1 PERTUSSIS 935 BORDETELLAPALELIRMVECSGK 1818 AAA22983.1 PERTUSSIS 936 BORDETELLA PFCFGKDLKRPG1819 AAA22983.1 PERTUSSIS 937 BORDETELLA PGSSPMEVMLRA 1820 AAA22983.1PERTUSSIS 938 BORDETELLA PGSSPMEVMLRAVF 1821 AAA22983.1 PERTUSSIS 939BORDETELLA PKQLTFEGK 1822 AAA22983.1 PERTUSSIS 940 BORDETELLAPLRMFLGPKQLT 1823 AAA22983.1 PERTUSSIS 941 BORDETELLA PTRMLVCGIAAK 1824AAA22983.1 PERTUSSIS 942 BORDETELLA PYVLVKTNMVVT 1825 AAA22983.1PERTUSSIS 943 BORDETELLA QLTFEGKPALELIRMVECSGKQDCP 1826 AAA22983.1PERTUSSIS 944 BORDETELLA QRPLRMFLGPKQ 1827 AAA22983.1 PERTUSSIS 945BORDETELLA RAVFMQQRPLRM 1828 AAA22983.1 PERTUSSIS 946 BORDETELLARMFLGPKQLTFE 1829 AAA22983.1 PERTUSSIS 947 BORDETELLA RMLVCGIAAKLG 1830AAA22983.1 PERTUSSIS 948 BORDETELLA RMVECSGKQDCP 1831 AAA22983.1PERTUSSIS 949 BORDETELLA SPDAHVPFCFGK 1832 AAA22983.1 PERTUSSIS 950BORDETELLA SSPMEVMLRAVF 1833 AAA22983.1 PERTUSSIS 951 BORDETELLASSPMEVMLRAVFMQQRPLRMFLGPK 1834 AAA22983.1 PERTUSSIS 952 BORDETELLASVAMKPYEVTPT 1835 AAA22983.1 PERTUSSIS 953 BORDETELLA VECSGKQDCP 1836AAA22983.1 PERTUSSIS 954 BORDETELLA VFMQQRPLRMFL 1837 AAA22983.1PERTUSSIS 955 BORDETELLA VFMQQRPLRMFLGPKQLTFEGKPAL 1838 AAA22983.1PERTUSSIS 956 BORDETELLA VKTNMVVTSVAM 1839 AAA22983.1 PERTUSSIS 957BORDETELLA VLVKTNMVVTSV 1840 AAA22983.1 PERTUSSIS 958 BORDETELLAVTPTRMLVCGIA 1841 AAA22983.1 PERTUSSIS 959 BORDETELLA VTSVAMKPYEVT 1842AAA22983.1 PERTUSSIS 960 BORDETELLA YEVTPTRMLVCG 1843 AAA22983.1PERTUSSIS 961 BORDETELLA YEVTPTRMLVCGIAAKLGAAASSPD 1844 AAA22983.1PERTUSSIS 962 BORDETELLA CASPYEGRYRDMYDALR 1845 P04979.1 PERTUSSIS 963BORDETELLA CAVFVRDGQSV 1846 P04979.1 PERTUSSIS 964 BORDETELLA CITTIYKTG1847 P04979.1 PERTUSSIS 965 BORDETELLA CPNGTRALTV 1848 P04979.1PERTUSSIS 966 BORDETELLA DALRRLLYMIYMSG 1849 P04979.1 PERTUSSIS 967RUBELLA VIRUS GNRGRGQRRDWSRAPPPPEERQETR 1850 P07566.1 STRAIN THERIEN S968 BORDETELLA GQPAADHYYSKVT 1851 P04979.1 PERTUSSIS 969 RUBELLA VIRUSGSPNCHGPDWASPVCQRHS 1852 ABD64214.1 970 MEASLES VIRUS HKSLSTNLDVTNSIEHQ1853 P08362.1 STRAIN EDMONSTON 971 BORDETELLA LFTQQGGAYGRC 1854 P04979.1PERTUSSIS 972 MEASLES VIRUS LIGLLAIAGIRLHRAAIYTAEI 1855 P08362.1 STRAINEDMONSTON 973 MEASLES PDTAADSELRRWIKY 1856 ABI54110.1 MORBILLIVIRUS 974RUBELLA VIRUS PNCHGPDWASPVCQRHS 1857 P07566.1 STRAIN THERIEN 975RUBELLA VIRUS QTPAPKPSRAPPQQPQPPRMQTGR 1858 ABD64216.1 976 RUBELLA VIRUSRAGLTAGASQSRRPRPPR 1859 CAA33016.1 VACCINE STRAIN RA27/3 977RUBELLA VIRUS RFGAPQAFLAGLLLAAVAVGTARA 1860 ABD64214.1 VACCINE STRAINRA27/3 978 BORDETELLA RGNAELQTYLRQITPG 1861 P04979.1 PERTUSSIS 979BORDETELLA RVHVSKEEQYYDYED 1862 P04979.1 PERTUSSIS 980 BORDETELLASIYGLYDGTYL 1863 P04979.1 PERTUSSIS 981 BORDETELLA SKVTATRLLASTNS 1864P04979.1 PERTUSSIS 982 BORDETELLA TQQGGAYGRCPNGTRA 1865 P04979.1PERTUSSIS 983 BORDETELLA VAPGIVIPPKAL 1866 P04979.1 PERTUSSIS 984BORDETELLA DSRPPEDVFQNGFTAWG 1867 ACI04548.1 PERTUSSIS 985 BORDETELLAEHRMQEAVEAERAGR 1868 ACI04548.1 PERTUSSIS 986 MEASLES VIRUSETCFQQACKGKIQALCENPEWA 1869 P08362.1 STRAIN EDMONSTON 987 BORDETELLAEYVDTYGDNAGRILAGALATYQ 1870 ACI04548.1 PERTUSSIS 988 BORDETELLAHRRIPPENIRRVTR 1871 ACI04548.1 PERTUSSIS 989 BORDETELLA MARQAESSE 1872ACI04548.1 PERTUSSIS 990 BORDETELLA MQEAVEAERAGR 1873 ACI04548.1PERTUSSIS 991 BORDETELLA SQQTRANPNPYTSRR 1874 ACI04548.1 PERTUSSIS 992BORDETELLA TRANPNPYTSRRSVASIVGTLVHG 1875 SRC280066 PERTUSSIS 993BORDETELLA TVYRYDSRPPED 1876 ACI04548.1 PERTUSSIS 994 MEASLES NDRNLLD1877 P10050.1 MORBILLIVIRUS 995 MEASLES NMEDEADQYFSHDDPISSDQSRFGW 1878P04851.1 MORBILLIVIRUS FENK 996 MEASLES SRASDARAAHLPTGTPLDID 1879P04851.1 MORBILLIVIRUS 997 BORDETELLA EDVFQNGFTAW 1880 ACI04548.1PERTUSSIS 998 CORYNEBACTERIUM AEGSSSVEYINNWEQAK 1881 AAV70486.1DIPHTHERIAE 999 CORYNEBACTERIUM GPIKNKMSESPNKT 1882 AAV70486.1DIPHTHERIAE 1000 MEASLES VIRUS GPKLTGALIGILSLFVESPGQLIQRITD 1883P10050.1 STRAIN HALLE DPDV 1001 CORYNEBACTERIUM GYQKTVDHTKVNSK 1884AAV70486.1 DIPHTHERIAE 1002 CORYNEBACTERIUM KTVDH 1885 AAV70486.1DIPHTHERIAE 1003 CORYNEBACTERIUM SESPNK 1886 AAV70486.1 DIPHTHERIAE 1004CORYNEBACTERIUM AEGSSSVEYINNWEQAKALS 1887 AAV70486.1 DIPHTHERIAE 1005CORYNEBACTERIUM AQAIPLVGELVDIGFAAYNF 1888 AAV70486.1 DIPHTHERIAE 1006CORYNEBACTERIUM ASRVVLSLPFAEGSSSVEYI 1889 AAV70486.1 DIPHTHERIAE 1007CORYNEBACTERIUM CINLDWDVIRDKTKTKIESL 1890 AAV70486.1 DIPHTHERIAE 1008CORYNEBACTERIUM CRAIDGDVTFCRPKSPVYVG 1891 AAV70486.1 DIPHTHERIAE 1009CORYNEBACTERIUM CRPKSPVYVGNGVHANLHVA 1892 AAV70486.1 DIPHTHERIAE 1010CORYNEBACTERIUM DAAGYSVDNENPLSGKAGGV 1893 AAV70486.1 DIPHTHERIAE 1011CORYNEBACTERIUM DKTKTKIESLKEHGPIKNKM 1894 AAV70486.1 DIPHTHERIAE 1012CORYNEBACTERIUM EEFIKRFGDGASRVVLSLPF 1895 AAV70486.1 DIPHTHERIAE 1013CORYNEBACTERIUM EKAKQYLEEFHQTALEHPEL 1896 AAV70486.1 DIPHTHERIAE 1014CORYNEBACTERIUM FHRSSSEKIHSNEISSDSIG 1897 AAV70486.1 DIPHTHERIAE 1015CORYNEBACTERIUM GADDVVDSSKSFVMENFSSY 1898 CAE11230.1 DIPHTHERIAE 1016CORYNEBACTERIUM GKRGQDAMYEYMAQACAGNR 1899 AAV70486.1 DIPHTHERIAE 1017CORYNEBACTERIUM GSVMGIADGAVHHNTEEIVA 1900 AAV70486.1 DIPHTHERIAE 1018CORYNEBACTERIUM GTQGNYDDDWKGFYSTDNKY 1901 AAV70486.1 DIPHTHERIAE 1019CORYNEBACTERIUM HDGYAVSWNTVEDSIIRTGF 1902 AAV70486.1 DIPHTHERIAE 1020CORYNEBACTERIUM HGTKPGYVDSIQKGIQKPKS 1903 AAV70486.1 DIPHTHERIAE 1021CORYNEBACTERIUM HQTALEHPELSELKTVTGTN 1904 AAV70486.1 DIPHTHERIAE 1022CORYNEBACTERIUM IQKGIQKPKSGTQGNYDDDW 1905 AAV70486.1 DIPHTHERIAE 1023CORYNEBACTERIUM KEHGPIKNKMSESPNKTVSE 1906 AAV70486.1 DIPHTHERIAE 1024CORYNEBACTERIUM KGFYSTDNKYDAAGYSVDNE 1907 AAV70486.1 DIPHTHERIAE 1025CORYNEBACTERIUM LDVNKSKTHISVNGRKIRMR 1908 AAV70486.1 DIPHTHERIAE 1026RUBELLA VIRUS MASTIPITMEDLQKALEA 1909 SRC265968 1027 CORYNEBACTERIUMNGVHANLHVAFHRSSSEKIH 1910 AAV70486.1 DIPHTHERIAE 1028 CORYNEBACTERIUMNNWEQAKALSVELEINFETR 1911 AAV70486.1 DIPHTHERIAE 1029 CORYNEBACTERIUMNPLSGKAGGVVKVTYPGLTK 1912 AAV70486.1 DIPHTHERIAE 1030 CORYNEBACTERIUMPVFAGANYAAWAVNVAQVID 1913 AAV70486.1 DIPHTHERIAE 1031 CORYNEBACTERIUMSELKTVTGTNPVFAGANYAA 1914 AAV70486.1 DIPHTHERIAE 1032 CORYNEBACTERIUMSESPNKTVSEEKAKQYLEEF 1915 AAV70486.1 DIPHTHERIAE 1033 CORYNEBACTERIUMSETADNLEKTTAALSILPGI 1916 AAV70486.1 DIPHTHERIAE 1034 CORYNEBACTERIUMSFVMENFSSYHGTKPGYVDS 1917 AAV70486.1 DIPHTHERIAE 1035 CORYNEBACTERIUMSNEISSDSIGVLGYQKTVDH 1918 AAV70486.1 DIPHTHERIAE 1036 CORYNEBACTERIUMSPGHKTQPFLHDGYAVSWNT 1919 AAV70486.1 DIPHTHERIAE 1037 CORYNEBACTERIUMSVNGRKIRMRCRAIDGDVTF 1920 AAV70486.1 DIPHTHERIAE 1038 CORYNEBACTERIUMTAALSILPGIGSVMGIADGA 1921 AAV70486.1 DIPHTHERIAE 1039 CORYNEBACTERIUMTAENTPLPIAGVLLPTIPGK 1922 AAV70486.1 DIPHTHERIAE 1040 CORYNEBACTERIUMTEPLMEQVGTEEFIKRFGDG 1923 AAV70486.1 DIPHTHERIAE 1041 CORYNEBACTERIUMTIKKELGLSLTEPLMEQVGT 1924 AAV70486.1 DIPHTHERIAE 1042 CORYNEBACTERIUMTKVNSKLSLFFEIKS 1925 AAV70486.1 DIPHTHERIAE 1043 CORYNEBACTERIUMVEDSIIRTGFQGESGHDIKI 1926 AAV70486.1 DIPHTHERIAE 1044 CORYNEBACTERIUMVELEINFETRGKRGQDAMYE 1927 AAV70486.1 DIPHTHERIAE 1045 CORYNEBACTERIUMVESIINLFQVVHNSYNRPAY 1928 AAV70486.1 DIPHTHERIAE 1046 CORYNEBACTERIUMVHNSYNRPAYSPGHKTQPFL 1929 AAV70486.1 DIPHTHERIAE 1047 CORYNEBACTERIUMVKVTYPGLTKVLALKVDNAE 1930 AAV70486.1 DIPHTHERIAE 1048 CORYNEBACTERIUMVLALKVDNAETIKKELGLSL 1931 AAV70486.1 DIPHTHERIAE 1049 CORYNEBACTERIUMVLGYQKTVDHTKVNSKLSLF 1932 AAV70486.1 DIPHTHERIAE 1050 CORYNEBACTERIUMVRRSVGSSLSCINLDWDVIR 1933 AAV70486.1 DIPHTHERIAE 1051 CORYNEBACTERIUMWAVNVAQVIDSETADNLEKT 1934 AAV70486.1 DIPHTHERIAE 1052 CORYNEBACTERIUMYMAQACAGNRVRRSVGSSLS 1935 AAV70486.1 DIPHTHERIAE 1053 BORDETELLAAKAPPAPKPAPQPGP 1936 ABO77783.1 PERTUSSIS 1054 BORDETELLA APKPAPQPGP1937 ABO77783.1 PERTUSSIS 1055 BORDETELLA APKPAPQPGPQPPQP 1938ABO77783.1 PERTUSSIS 1056 MEASLES VIRUS AQTRTPLQCTMTEIF 1939 P04851.1STRAIN EDMONSTON 1057 MEASLES VIRUS ASRGTNMEDEADQYFSHDD 1940 P04851.1STRAIN EDMONSTON 1058 BORDETELLA ATIRR 1941 ABO77783.1 PERTUSSIS 1059BORDETELLA DNRAG 1942 ABO77783.1 PERTUSSIS 1060 BORDETELLAEAPAPQPPAGRELSA 1943 ABO77783.1 PERTUSSIS 1061 MEASLES VIRUSEMVRRSAGKVSSTLASELGI 1944 P04851.1 STRAIN EDMONSTON 1062 BORDETELLAGASEL 1945 ABO77783.1 PERTUSSIS 1063 BORDETELLA GDALAGGAVP 1946AAA22980.1 PERTUSSIS 1064 BORDETELLA GDAPAGGAVP 1947 ABO77783.1PERTUSSIS 1065 BORDETELLA GDTWDDD 1948 ABO77783.1 PERTUSSIS 1066BORDETELLA GERQH 1949 ABO77783.1 PERTUSSIS 1067 BORDETELLA GGAVP 1950ABO77783.1 PERTUSSIS 1068 BORDETELLA GGFGP 1951 P14283.3 PERTUSSIS 1069BORDETELLA GGFGPGGFGP 1952 BAF35031.1 PERTUSSIS 1070 BORDETELLAGGFGPVLDGW 1953 ABO77783.1 PERTUSSIS 1071 MEASLES VIRUSGGKEDRRVKQSRGEARESYR 1954 P04851.1 STRAIN EDMONSTON 1072 BORDETELLAGILLEN 1955 ABO77783.1 PERTUSSIS 1073 BORDETELLA GIRRFL 1956 ABO77783.1PERTUSSIS 1074 MEASLES VIRUS HDDPISSDQSRFGWFENKEI 1957 P04851.1 STRAINEDMONSTON 1075 MEASLES VIRUS HEFAGELSTLESLMNLY 1958 P04851.1 STRAINEDMONSTON 1076 BORDETELLA HLGGLAGY 1959 ABO77783.1 PERTUSSIS 1077MEASLES VIRUS HTTEDKISRAVGPRQAQVSFL 1960 P04851.1 STRAIN EDMONSTON 1078MEASLES VIRUS ICDIDTYIVEAGLASFILTI 1961 P04851.1 STRAIN EDMONSTON 1079MEASLES VIRUS IIIVPIPGDSSITTRSRLLD 1962 P04851.1 STRAIN EDMONSTON 1080MEASLES VIRUS ILDIKRTPGNKPRIAEMICD 1963 P04851.1 STRAIN EDMONSTON 1081BORDETELLA KALLYR 1964 ABO77783.1 PERTUSSIS 1082 MEASLES VIRUSKPPITSGSGGAIRGIKHIII 1965 P04851.1 STRAIN EDMONSTON 1083 BORDETELLALAGSGL 1966 ABO77783.1 PERTUSSIS 1084 MEASLES VIRUS LGITAEDARLVSEIAMHTTE1967 P04851.1 STRAIN EDMONSTON 1085 MEASLES VIRUS LGTILAQIWVLLAKAVTA1968 P04851.1 STRAIN EDMONSTON 1086 MEASLES VIRUS LPTGTPLDIDTASESSQD1969 P04851.1 STRAIN EDMONSTON 1087 MEASLES VIRUS MATLLRSLALFKRNKDK 1970P04851.1 STRAIN EDMONSTON 1088 BORDETELLA PAPQPP 1971 ABO77783.1PERTUSSIS 1089 MEASLES VIRUS PDTAADSELRRWIKYTQQRR 1972 P04851.1 STRAINEDMONSTON 1090 MEASLES VIRUS PKLTGALIGILSLFVESPGQ 1973 P04851.1 STRAINEDMONSTON 1091 BORDETELLA PQP 1974 ABO77783.1 PERTUSSIS 1092 BORDETELLAPQPGP 1975 ABO77783.1 PERTUSSIS 1093 BORDETELLA PQPGPQPPQPPQPQP 1976ABO77783.1 PERTUSSIS 1094 MEASLES VIRUS QDPQDSRRSAEPLLSCKPWQ 1977P04851.1 STRAIN EDMONSTON 1095 MEASLES VIRUS QRRVVGEFRLERKWLDVVR 1978P04851.1 STRAIN EDMONSTON 1096 BORDETELLA RELSA 1979 ABO77783.1PERTUSSIS 1097 BORDETELLA RFAPQ 1980 ABO77783.1 PERTUSSIS 1098MEASLES VIRUS SIQNKFSAGSYPLLWSYAMG 1981 P04851.1 STRAIN EDMONSTON 1099BORDETELLA SITLQAGAH 1982 ABO77783.1 PERTUSSIS 1100 BORDETELLA SLQPED1983 ABO77783.1 PERTUSSIS 1101 BORDETELLA SNALSKRL 1984 ABO77783.1PERTUSSIS 1102 MEASLES VIRUS SPGQLIQRITDDPDVSIRLL 1985 P04851.1 STRAINEDMONSTON 1103 BORDETELLA TELPSIPG 1986 ABO77783.1 PERTUSSIS 1104BORDETELLA TFTLANK 1987 ABO77783.1 PERTUSSIS 1105 BORDETELLA TWDDD 1988ABO77783.1 PERTUSSIS 1106 MEASLES VIRUS VSFLQGDQSENELPRLGGKE 1989P04851.1 STRAIN EDMONSTON 1107 MEASLES VIRUS WQESRKNKAQTRTPLQC 1990P04851.1 STRAIN EDMONSTON 1108 MEASLES VIRUS YAMGVGVELENSMGGLNFGR 1991P04851.1 STRAIN EDMONSTON 1109 MEASLES VIRUS YQQMGKPAPYMVNLENSI 1992P04851.1 STRAIN EDMONSTON 1110 MEASLES VIRUS MTRSSHQSLVIKLMP 1993P69355.1 STRAIN HALLE 1111 MEASLES VIRUS PIRDALNAMTQNIRP 1994 P69355.1STRAIN HALLE 1112 RUBELLA VIRUS ALLNTPPPYQVSCGGESDRASAGH 1995 CAA28880.1STRAIN M33 1113 RUBELLA VIRUS GLGSPNCHGPDWASPVCQRHS 1996 P07566.1STRAIN THERIEN 1114 RUBELLA VIRUS GLGSPNCHGPDWASPVCQRHSPDCS 1997P07566.1 STRAIN THERIEN RLV 1115 RUBELLA VIRUS NYTGNQQSRWGLGSPNCHGPDWASP1998 P07566.1 STRAIN THERIEN V 1116 RUBELLA VIRUS TLPQPPCAHGQHYGHHHHQL1999 P07566.1 STRAIN THERIEN 1117 RUBELLA VIRUSTVRVKFHTETRTVWQLSVAGVSCNV 2000 P07566.1 STRAIN THERIEN T 1118RUBELLA VIRUS ASYFNPGGSYYKQYH 2001 BAA28178.1 1119 RUBELLA VIRUSFALASYVQHPHKTVR 2002 BAA28178.1 1120 RUBELLA VIRUS GGESDRASARVIDPAAQSFTG2003 BAA28178.1 1121 RUBELLA VIRUS GPGEVWVTPVIGSQARKC 2004 BAA28178.11122 RUBELLA VIRUS GSQARKCGLHIRAG 2005 BAA28178.1 1123 RUBELLA VIRUSLVGATPERPRLRLVDADDPLLRTAP 2006 BAA28178.1 1124 RUBELLA VIRUSTAPGPGEVWVTPVI 2007 BAA28178.1 1125 MEASLES FIVLSIAYPTLSEIK 2008AAL29688.1 MORBILLIVIRUS 1126 MEASLES VIRUS ETCFQQACKGKIQALCENPEWAPLK2009 P08362.1 STRAIN DNRIPSY EDMONSTON- ZAGREB 1127 MEASLES LPRLGGKEDR2010 P04851.1 MORBILLIVIRUS 1128 MEASLES MSKTEWNASQ 2011 SRC280080MORBILLIVIRUS 1129 MEASLES SRFGWFENKE 2012 P04851.1 MORBILLIVIRUS 1130RUBELLA VIRUS EACVTSWLWSEGEGAVFYRVDLHFI 2013 CAA28880.1 NLGT 1131RUBELLA VIRUS MDFWCVEHDRPPPATPTSLTT 2014 CAA33016.1 1132 RUBELLA VIRUSPFLGHDGHHGGTLRVGQHHRNASDV 2015 CAA33016.1 1133 RUBELLA VIRUSRVKFHTETRTVWQLSVAGVSC 2016 BAA19893.1 1134 MEASLES AEEQARHVKNGLE 2017ABO69699.1 MORBILLIVIRUS 1135 MEASLES ESPQEISKHQALG 2018 SRC280080MORBILLIVIRUS 1136 MEASLES GVGVELENSMGGLNF 2019 ABI54110.1 MORBILLIVIRUS1137 MEASLES IKGANDLAKFHQMLMKIIMK 2020 ABO69699.1 MORBILLIVIRUS 1138MEASLES MSKTLHAQLGFKKT 2021 ABK40528.1 MORBILLIVIRUS 1139 MEASLESNASGLSRPSPSAH 2022 BAE98296.1 MORBILLIVIRUS 1140 MEASLES VRVIDPSLGDRKDE2023 SRC280148 MORBILLIVIRUS 1141 BORDETELLA DLSDGDLLV 2024 AAC31207.1PERTUSSIS 1142 BORDETELLA EAERAGRGTG 2025 ACI04548.1 PERTUSSIS 1143BORDETELLA YRYDSRPPEDV 2026 ACI04548.1 PERTUSSIS 1144 BORDETELLAYVDTYGDNAG 2027 ACI04548.1 PERTUSSIS 1145 MEASLES SFSYFYPFR 2028CAB43772.1 MORBILLIVIRUS 1146 MUMPS DIFIVSPR 2029 ADF49557.1 RUBULAVIRUS1147 MEASLES QDSRRSADALLRLQAMAGISEEQGS 2030 CAA59302.1 MORBILLIVIRUSDTDTPIVYNDRN 1148 MEASLES SAEALLRLQA 2031 BAH22350.1 MORBILLIVIRUS 1149MEASLES RIVINREHL 2032 BAB39835.1 MORBILLIVIRUS 1150 MEASLES VIRUS IPRFK2033 BAB39848.1 GENOTYPEA 1151 BORDETELLA AAALSPMEI 2034 P15318.2PERTUSSIS 1152 BORDETELLA AAASVVGAPV 2035 P15318.2 PERTUSSIS 1153BORDETELLA AALGRQDSI 2036 P15318.2 PERTUSSIS 1154 BORDETELLA AAQRLVHAIA2037 P15318.2 PERTUSSIS 1155 BORDETELLA AAVEAAEL 2038 P15318.2 PERTUSSIS1156 BORDETELLA AGANVLNGL 2039 P15318.2 PERTUSSIS 1157 BORDETELLAAGYANAAD 2040 P15318.2 PERTUSSIS 1158 BORDETELLA AGYEQFEFRV 2041P15318.2 PERTUSSIS 1159 BORDETELLA AITGNADNL 2042 P15318.2 PERTUSSIS1160 BORDETELLA AKEKNATLM 2043 P15318.2 PERTUSSIS 1161 BORDETELLAAKGVFLSL 2044 P15318.2 PERTUSSIS 1162 BORDETELLA APHEYGFGI 2045 P15318.2PERTUSSIS 1163 BORDETELLA ARQGNDLEI 2046 P15318.2 PERTUSSIS 1164BORDETELLA ASVVGAPV 2047 P15318.2 PERTUSSIS 1165 BORDETELLA ATLMFRLV2048 P15318.2 PERTUSSIS 1166 BORDETELLA AVAAAQRL 2049 P15318.2 PERTUSSIS1167 BORDETELLA DAGANVLNGL 2050 P15318.2 PERTUSSIS 1168 BORDETELLADALLAQLYR 2051 P15318.2 PERTUSSIS 1169 BORDETELLA DANGVLKHSI 2052P15318.2 PERTUSSIS 1170 BORDETELLA DGDMNIGVI 2053 P15318.2 PERTUSSIS1171 BORDETELLA DHVKNIENL 2054 P15318.2 PERTUSSIS 1172 BORDETELLADIDMFAIM 2055 P15318.2 PERTUSSIS 1173 BORDETELLA DMFAIMPHL 2056 P15318.2PERTUSSIS 1174 BORDETELLA DNVRNVENV 2057 P15318.2 PERTUSSIS 1175BORDETELLA DNVRNVENVI 2058 P15318.2 PERTUSSIS 1176 BORDETELLA DTVDYSAM2059 P15318.2 PERTUSSIS 1177 BORDETELLA DTVDYSAMI 2060 P15318.2PERTUSSIS 1178 BORDETELLA DYLRQAGL 2061 P15318.2 PERTUSSIS 1179BORDETELLA DYYDNVRNV 2062 P15318.2 PERTUSSIS 1180 BORDETELLA EFTTFVEI2063 P15318.2 PERTUSSIS 1181 BORDETELLA EFTTFVEIV 2064 P15318.2PERTUSSIS 1182 BORDETELLA EGYVFYEN 2065 P15318.2 PERTUSSIS 1183BORDETELLA ENVQYRHV 2066 P15318.2 PERTUSSIS 1184 BORDETELLA EQLANSDGL2067 P15318.2 PERTUSSIS 1185 BORDETELLA FGVGYGHDTI 2068 P15318.2PERTUSSIS 1186 BORDETELLA FSPDVLETVP 2069 P15318.2 PERTUSSIS 1187BORDETELLA FSVDHVKNI 2070 P15318.2 PERTUSSIS 1188 BORDETELLA GDDTYLFGV2071 P15318.2 PERTUSSIS 1189 BORDETELLA GDDVFLQDL 2072 P15318.2PERTUSSIS 1190 BORDETELLA GEDGNDIFL 2073 P15318.2 PERTUSSIS 1191BORDETELLA GERFNVRKQL 2074 P15318.2 PERTUSSIS 1192 BORDETELLA GGAGNDTLV2075 P15318.2 PERTUSSIS 1193 BORDETELLA GGDDFEAV 2076 P15318.2 PERTUSSIS1194 BORDETELLA GKSEFTTFV 2077 P15318.2 PERTUSSIS 1195 BORDETELLAGKSLFDDGL 2078 P15318.2 PERTUSSIS 1196 BORDETELLA GNADNLKSV 2079P15318.2 PERTUSSIS 1197 BORDETELLA GQLVEVDTL 2080 P15318.2 PERTUSSIS1198 BORDETELLA GRSKFSPDV 2081 P15318.2 PERTUSSIS 1199 BORDETELLAGSSAYDTV 2082 P15318.2 PERTUSSIS 1200 BORDETELLA GTVEKWPAL 2083 P15318.2PERTUSSIS 1201 BORDETELLA GVDYYDNV 2084 P15318.2 PERTUSSIS 1202BORDETELLA GYEQFEFRV 2085 P15318.2 PERTUSSIS 1203 BORDETELLA HAVGAQDVV2086 P15318.2 PERTUSSIS 1204 BORDETELLA IAAGRIGLGI 2087 P15318.2PERTUSSIS 1205 BORDETELLA IGDAQANTL 2088 P15318.2 PERTUSSIS 1206BORDETELLA IGLGILADL 2089 P15318.2 PERTUSSIS 1207 BORDETELLA IGNAAGIPL2090 P15318.2 PERTUSSIS 1208 BORDETELLA IGTSMKDVL 2091 P15318.2PERTUSSIS 1209 BORDETELLA IGVITDFEL 2092 P15318.2 PERTUSSIS 1210BORDETELLA IPLTADIDM 2093 P15318.2 PERTUSSIS 1211 BORDETELLA ISKSALEL2094 P15318.2 PERTUSSIS 1212 BORDETELLA ITGNADNL 2095 P15318.2 PERTUSSIS1213 BORDETELLA KIFVVSAT 2096 P15318.2 PERTUSSIS 1214 BORDETELLAKQLNNANVYR 2097 P15318.2 PERTUSSIS 1215 BORDETELLA KVIGNAAGI 2098P15318.2 PERTUSSIS 1216 BORDETELLA LAKVVSQL 2099 P15318.2 PERTUSSIS 1217BORDETELLA LANDYARKI 2100 P15318.2 PERTUSSIS 1218 BORDETELLA LDYLRQAGL2101 P15318.2 PERTUSSIS 1219 BORDETELLA LGKGFASL 2102 P15318.2 PERTUSSIS1220 BORDETELLA LGKGFASLM 2103 P15318.2 PERTUSSIS 1221 BORDETELLALGVDYYDN 2104 P15318.2 PERTUSSIS 1222 BORDETELLA LGVDYYDNV 2105 P15318.2PERTUSSIS 1223 BORDETELLA LKHSIKLDVI 2106 P15318.2 PERTUSSIS 1224BORDETELLA LQAGYIPV 2107 P15318.2 PERTUSSIS 1225 BORDETELLA LQLTGGTVE2108 P15318.2 PERTUSSIS 1226 BORDETELLA LSAAVFGL 2109 P15318.2 PERTUSSIS1227 BORDETELLA LSLGKGFASL 2110 P15318.2 PERTUSSIS 1228 BORDETELLALSPMEIYGL 2111 P15318.2 PERTUSSIS 1229 BORDETELLA NAHDNFLAGG 2112P15318.2 PERTUSSIS 1230 BORDETELLA NANVYREGV 2113 P15318.2 PERTUSSIS1231 BORDETELLA NDTLYGGL 2114 P15318.2 PERTUSSIS 1232 BORDETELLANGLAGNDVL 2115 P15318.2 PERTUSSIS 1233 BORDETELLA NNANVYREGV 2116P15318.2 PERTUSSIS 1234 BORDETELLA NTVSYAAL 2117 P15318.2 PERTUSSIS 1235BORDETELLA NVLRNIENAV 2118 P15318.2 PERTUSSIS 1236 BORDETELLA PALTFITPL2119 P15318.2 PERTUSSIS 1237 BORDETELLA PETSNVLRNI 2120 P15318.2PERTUSSIS 1238 BORDETELLA PMEIYGLV 2121 P15318.2 PERTUSSIS 1239BORDETELLA PQAYFEKNL 2122 P15318.2 PERTUSSIS 1240 BORDETELLA PVNPNLSKL2123 P15318.2 PERTUSSIS 1241 BORDETELLA QAGWNASSV 2124 P15318.2PERTUSSIS 1242 BORDETELLA QAGWNASSVI 2125 P15318.2 PERTUSSIS 1243BORDETELLA QDAANAGNL 2126 P15318.2 PERTUSSIS 1244 BORDETELLA QDAANAGNLL2127 P15318.2 PERTUSSIS 1245 BORDETELLA QDSGYDSL 2128 P15318.2 PERTUSSIS1246 BORDETELLA QQSHYADQL 2129 P15318.2 PERTUSSIS 1247 BORDETELLARALQGAQAV 2130 P15318.2 PERTUSSIS 1248 BORDETELLA RGGLGLDTL 2131P15318.2 PERTUSSIS 1249 BORDETELLA RKQLNNANV 2132 P15318.2 PERTUSSIS1250 BORDETELLA RQDSGYDSL 2133 P15318.2 PERTUSSIS 1251 BORDETELLARQFRYDGDM 2134 P15318.2 PERTUSSIS 1252 BORDETELLA RSKFSPDVL 2135P15318.2 PERTUSSIS 1253 BORDETELLA SAGAAAGAL 2136 P15318.2 PERTUSSIS1254 BORDETELLA SAHWGQRAL 2137 P15318.2 PERTUSSIS 1255 BORDETELLASAMIHPGRI 2138 P15318.2 PERTUSSIS 1256 BORDETELLA SAMIHPGRIV 2139P15318.2 PERTUSSIS 1257 BORDETELLA SAYDTVSGI 2140 P15318.2 PERTUSSIS1258 BORDETELLA SAYGYEGD 2141 P15318.2 PERTUSSIS 1259 BORDETELLASGGAGDDVL 2142 P15318.2 PERTUSSIS 1260 BORDETELLA SGLQVAGA 2143 P15318.2PERTUSSIS 1261 BORDETELLA SGYDSLDGV 2144 P15318.2 PERTUSSIS 1262BORDETELLA SLLTGALNGI 2145 P15318.2 PERTUSSIS 1263 BORDETELLA SPMEIYGL2146 P15318.2 PERTUSSIS 1264 BORDETELLA SQMLTRGQL 2147 P15318.2PERTUSSIS 1265 BORDETELLA SSAYDTVSGI 2148 P15318.2 PERTUSSIS 1266BORDETELLA SSLAHGHTA 2149 P15318.2 PERTUSSIS 1267 BORDETELLA SSVTSGDSV2150 P15318.2 PERTUSSIS 1268 BORDETELLA SVIGVQTTEI 2151 P15318.2PERTUSSIS 1269 BORDETELLA TNTVSYAAL 2152 P15318.2 PERTUSSIS 1270BORDETELLA TSLIAEGV 2153 P15318.2 PERTUSSIS 1271 BORDETELLA TSLLTGAL2154 P15318.2 PERTUSSIS 1272 BORDETELLA TVPASPGL 2155 P15318.2 PERTUSSIS1273 BORDETELLA VAKEKNATL 2156 P15318.2 PERTUSSIS 1274 BORDETELLAVAPHEYGFGI 2157 P15318.2 PERTUSSIS 1275 BORDETELLA VAVVTSLL 2158P15318.2 PERTUSSIS 1276 BORDETELLA VFYENRAYG 2159 P15318.2 PERTUSSIS1277 BORDETELLA VFYENRAYGV 2160 P15318.2 PERTUSSIS 1278 BORDETELLAVITDFELEV 2161 P15318.2 PERTUSSIS 1279 BORDETELLA VNPHSTSL 2162 P15318.2PERTUSSIS 1280 BORDETELLA VNPHSTSLI 2163 P15318.2 PERTUSSIS 1281BORDETELLA VNPNLSKL 2164 P15318.2 PERTUSSIS 1282 BORDETELLA VNPNLSKLF2165 P15318.2 PERTUSSIS 1283 BORDETELLA VQQPIIEKL 2166 P15318.2PERTUSSIS 1284 BORDETELLA VQYRHVEL 2167 P15318.2 PERTUSSIS 1285BORDETELLA VSIAAAASV 2168 P15318.2 PERTUSSIS 1286 BORDETELLA VSIAAAASVV2169 P15318.2 PERTUSSIS 1287 BORDETELLA VTSLLTGAL 2170 P15318.2PERTUSSIS 1288 BORDETELLA VVLANASRI 2171 P15318.2 PERTUSSIS 1289BORDETELLA WPALNLFSV 2172 P15318.2 PERTUSSIS 1290 BORDETELLA WVRKASAL2173 P15318.2 PERTUSSIS 1291 BORDETELLA YAVQYRRKGG 2174 P15318.2PERTUSSIS 1292 BORDETELLA YGGLGDDTL 2175 P15318.2 PERTUSSIS 1293BORDETELLA YGLVQQSHYA 2176 P15318.2 PERTUSSIS 1294 BORDETELLA YGYEGDALL2177 P15318.2 PERTUSSIS 1295 BORDETELLA YIPVNPNL 2178 P15318.2 PERTUSSIS1296 BORDETELLA YSAMIHPGRI 2179 P15318.2 PERTUSSIS 1297 BORDETELLAYSQTGAHAGI 2180 P15318.2 PERTUSSIS 1298 CORYNEBACTERIUMAYNFVESIINLFQVVHNSYN 2181 CAE11230.1 DIPHTHERIAE 1299 BORDETELLA SGTTIK2182 BAF35031.1 PERTUSSIS 1300 BORDETELLA RGHTLESAEGRKIFG 2183AAA22974.1 PERTUSSIS 1301 BORDETELLA AGAMTVRDVAAAADLALQAGDA 2184AAA22974.1 PERTUSSIS 1302 BORDETELLA AGAMTVRDVAAAADLALQAGDAL 2185AAA22974.1 PERTUSSIS 1303 BORDETELLA ALAAVLVNPHIFTRIGAAQTSLADGA 2186AAA22974.1 PERTUSSIS AGPA 1304 BORDETELLA ALSIDSMTALGA 2187 AAA22974.1PERTUSSIS 1305 BORDETELLA DLSAARGADISGEGR 2188 AAA22974.1 PERTUSSIS 1306BORDETELLA DQNRYEYIWGLY 2189 AAA22974.1 PERTUSSIS 1307 BORDETELLADYTVSADAIALA 2190 AAA22974.1 PERTUSSIS 1308 BORDETELLA GPIVVEAGELVSHAGG2191 AAA22974.1 PERTUSSIS 1309 BORDETELLA GRPEGLKIGAHSATSVSGSFDAL 2192AAA22974.1 PERTUSSIS 1310 BORDETELLA ITVTSRGGFDNEGKMESNK 2193 AAA22974.1PERTUSSIS 1311 BORDETELLA LDQNRYEYIWGLYP 2194 AAA22974.1 PERTUSSIS 1312BORDETELLA LSAARGADISG 2195 AAA22974.1 PERTUSSIS 1313 BORDETELLANKIRLMGPLQ 2196 AAA22974.1 PERTUSSIS 1314 BORDETELLA NKLGRIRAGEDM 2197AAA22974.1 PERTUSSIS 1315 BORDETELLA NKLGRIRAGEDMHLDAPRIE 2198AAA22974.1 PERTUSSIS 1316 BORDETELLA PHLRNTGQVVAG 2199 AAA22974.1PERTUSSIS 1317 BORDETELLA QVDLHDLSAARGADISG 2200 AAA22974.1 PERTUSSIS1318 BORDETELLA RDVAAAADLALQ 2201 AAA22974.1 PERTUSSIS 1319 BORDETELLASAARGADISGEG 2202 AAA22974.1 PERTUSSIS 1320 BORDETELLA TKGEMQIAGKGGGSP2203 AAA22974.1 PERTUSSIS 1321 BORDETELLA TVSADAIALAAQ 2204 AAA22974.1PERTUSSIS 1322 BORDETELLA VVAGHDIHI 2205 AAA22974.1 PERTUSSIS 1323BORDETELLA PSGPNHTKVVQLPKISKNALKANG 2206 CAD12823.1 PERTUSSIS 1324RUBELLA VIRUS LVGATPERPRLRLVDADDPLLRTAPG 2207 BAA19902.1 PGEVWVTPVIGSQAR1325 RUBELLA VIRUS QQSRWGLGSPNCHGPDWASPVCQRH 2208 BAA19902.1 SP 1326BORDETELLA AGEAMVLVYYESIAYSF 2209 ACI04548.1 PERTUSSIS 1327 BORDETELLAGGVGLASTLWYAESNALSKRLGEL 2210 AAZ74322.1 PERTUSSIS 1328 BORDETELLAGTLVRIAPVIGACMARQA 2211 ACI04548.1 PERTUSSIS 1329 BORDETELLAIRRVTRVYHNGITGETTT 2212 ACI04548.1 PERTUSSIS 1330 BORDETELLAIVKTGERQHGIHIQGSDP 2213 AAZ74322.1 PERTUSSIS 1331 BORDETELLAIVKTGERQHGIHIQGSDPGGVRTA 2214 AAZ74338.1 PERTUSSIS 1332 BORDETELLALRDTNVTAVPASGAPAAVSVLGAS 2215 AAZ74338.1 PERTUSSIS 1333 BORDETELLAPEAPAPQPPAGRELSAAANAAVNT 2216 AAZ74322.1 PERTUSSIS 1334 BORDETELLAAAADFAHAE 2217 WP_019247158.1 PERTUSSIS 1335 BORDETELLA AAAEVAGAL 2218WP_019249248.1 PERTUSSIS 1336 BORDETELLA AAESTFESY 2219 WP_019247158.1PERTUSSIS 1337 BORDETELLA AAGFDPEVQ 2220 WP_019248145.1 PERTUSSIS 1338BORDETELLA AALGRGHSL 2221 AGS56996.1 PERTUSSIS 1339 BORDETELLA AAMQGAVVH2222 AGT50936.1 PERTUSSIS 1340 BORDETELLA AAPAAHADW 2223 AGS56996.1PERTUSSIS 1341 BORDETELLA AAQATVVQR 2224 WP_019247158.1 PERTUSSIS 1342BORDETELLA AARVAGDNY 2225 WP_019249248.1 PERTUSSIS 1343 BORDETELLAAAVALLNKL 2226 WP_019249248.1 PERTUSSIS 1344 BORDETELLA ADDPPATVY 2227AAW72734.1 PERTUSSIS 1345 BORDETELLA AEAGRFKVL 2228 AGS56996.1 PERTUSSIS1346 BORDETELLA AEATQLVTA 2229 WP_019247158.1 PERTUSSIS 1347 BORDETELLAAEGGATLGA 2230 WP_019249248.1 PERTUSSIS 1348 BORDETELLA AEHGEVSIQ 2231WP_019249248.1 PERTUSSIS 1349 BORDETELLA AEIAFYPKE 2232 WP_019249248.1PERTUSSIS 1350 BORDETELLA AEKVTTPAV 2233 WP_019247158.1 PERTUSSIS 1351BORDETELLA AELQTYLRQ 2234 1BCP_C PERTUSSIS 1352 BORDETELLA AEQSLIEVG2235 WP_019249248.1 PERTUSSIS 1353 BORDETELLA AESNALSKR 2236 AGS56996.1PERTUSSIS 1354 BORDETELLA AESSEAMAA 2237 AFK26302.1 PERTUSSIS 1355BORDETELLA AEVKVGYRA 2238 WP_019247158.1 PERTUSSIS 1356 BORDETELLAAEVTDTSPS 2239 WP_019249248.1 PERTUSSIS 1357 BORDETELLA AGKSLKKKN 2240WP_019247158.1 PERTUSSIS 1358 BORDETELLA AGLAGPSAV 2241 WP_019249248.1PERTUSSIS 1359 BORDETELLA AIRVGRGAR 2242 AGT50936.1 PERTUSSIS 1360BORDETELLA ALAAIASAA 2243 WP_019248145.1 PERTUSSIS 1361 BORDETELLAALADVPYVL 2244 AAA22983.1 PERTUSSIS 1362 BORDETELLA ALANDGTIV 2245WP_019248658.1 PERTUSSIS 1363 BORDETELLA ALGRGHSLY 2246 AGS56996.1PERTUSSIS 1364 BORDETELLA ALILAASPV 2247 WP_019248658.1 PERTUSSIS 1365BORDETELLA ALMLACTGL 2248 AAA22974.1 PERTUSSIS 1366 BORDETELLA AMQGAVVHL2249 AGT50936.1 PERTUSSIS 1367 BORDETELLA AMTHLSPAL 2250 AAA22983.1PERTUSSIS 1368 BORDETELLA AMYGKHITL 2251 WP_019249248.1 PERTUSSIS 1369BORDETELLA ANEANALLW 2252 WP_019249248.1 PERTUSSIS 1370 BORDETELLAAPLSITLQA 2253 AGT50936.1 PERTUSSIS 1371 BORDETELLA APNALAWAL 2254AAA22974.1 PERTUSSIS 1372 BORDETELLA APPAPKPAP 2255 AGS56996.1 PERTUSSIS1373 BORDETELLA APPGAGFIY 2256 1BCP_C PERTUSSIS 1374 BORDETELLAAPQAAPLSI 2257 AGT50936.1 PERTUSSIS 1375 BORDETELLA PRIENTAK 2258WP_019249248.1 PERTUSSIS 1376 BORDETELLA AQGKALLYR 2259 AGT50936.1PERTUSSIS 1377 BORDETELLA AQITSYVGF 2260 WP_019248658.1 PERTUSSIS 1378BORDETELLA AQLEVRGQR 2261 WP_019249248.1 PERTUSSIS 1379 BORDETELLAAQQLKQADR 2262 WP_019247699.1 PERTUSSIS 1380 BORDETELLA AQVTVAGRY 2263WP_019249248.1 PERTUSSIS 1381 BORDETELLA ARRSRVRAL 2264 N_882284.1PERTUSSIS 1382 BORDETELLA ASPRRARRA 2265 WP_019249248.1 PERTUSSIS 1383BORDETELLA ASSPDAHVP 2266 AAA22983.1 PERTUSSIS 1384 BORDETELLA ASVSNPGTF2267 WP_019249248.1 PERTUSSIS 1385 BORDETELLA ATWNFQSTY 2268WP_019249248.1 PERTUSSIS 1386 BORDETELLA ATYIADSGF 2269 AGS56996.1PERTUSSIS 1387 BORDETELLA AVAAPAVGA 2270 WP_019249248.1 PERTUSSIS 1388BORDETELLA AVFMQQRPL 2271 AAA22983.1 PERTUSSIS 1389 BORDETELLA AVLVNPHIF2272 WP_019249248.1 PERTUSSIS 1390 BORDETELLA CFGKDLKRP 2273 AAA22983.1PERTUSSIS 1391 BORDETELLA CPSSLGNGV 2274 WP_019248145.1 PERTUSSIS 1392BORDETELLA DAGHEHDTW 2275 AAA22984.1 PERTUSSIS 1393 BORDETELLA DAKHDLTVT2276 WP_019249248.1 PERTUSSIS 1394 BORDETELLA DASGQHRLW 2277 AGS56996.1PERTUSSIS 1395 BORDETELLA DATFETYAL 2278 YP_006628018.1 PERTUSSIS 1396BORDETELLA DATFQTYAL 2279 1BCP_C PERTUSSIS 1397 BORDETELLA DATLVGAKF2280 WP_019247158.1 PERTUSSIS 1398 BORDETELLA DDEVDVSGR 2281 AAA22974.1PERTUSSIS 1399 BORDETELLA DENGKPQTY 2282 WP_019247158.1 PERTUSSIS 1400BORDETELLA DGPPSRPTT 2283 WP_019247158.1 PERTUSSIS 1401 BORDETELLADHLTGRSCQ 2284 AAW72734.1 PERTUSSIS 1402 BORDETELLA DNEGKMESN 2285WP_019249248.1 PERTUSSIS 1403 BORDETELLA DPPATVYRY 2286 AAW72734.1PERTUSSIS 1404 BORDETELLA EATEGDATL 2287 WP_019247158.1 PERTUSSIS 1405BORDETELLA EATQQAAGF 2288 WP_019248145.1 PERTUSSIS 1406 BORDETELLAECSGKQDCP 2289 AAA22983.1 PERTUSSIS 1407 BORDETELLA EGGKLRGKD 2290AAA22974.1 PERTUSSIS 1408 BORDETELLA EGKMESNKD 2291 WP_019249248.1PERTUSSIS 1409 BORDETELLA EHRMQEAVE 2292 AAW72734.1 PERTUSSIS 1410BORDETELLA EKRLDIDDA 2293 WP_019249248.1 PERTUSSIS 1411 BORDETELLAEPASANTLL 2294 AGS56996.1 PERTUSSIS 1412 BORDETELLA EPQAELAVF 2295AGS56996.1 PERTUSSIS 1413 BORDETELLA EPVKLTLTG 2296 AGT50936.1 PERTUSSIS1414 BORDETELLA ESAEGRKIF 2297 WP_019249248.1 PERTUSSIS 1415 BORDETELLAESYSESHNF 2298 WP_019247158.1 PERTUSSIS 1416 BORDETELLA ETFCITTIY 22991BCP_C PERTUSSIS 1417 BORDETELLA EVAGALELS 2300 WP_019249248.1 PERTUSSIS1418 BORDETELLA EVAKVEVVP 2301 WP_019247158.1 PERTUSSIS 1419 BORDETELLAEVDGIIQEF 2302 WP_019249248.1 PERTUSSIS 1420 BORDETELLA EVRADNNFY 2303WP_019248344.1 PERTUSSIS 1421 BORDETELLA FAILSSTTE 2304 WP_019247158.1PERTUSSIS 1422 BORDETELLA FAISAYALK 2305 AAA22984.1 PERTUSSIS 1423BORDETELLA FALYDGTYL 2306 AFK26303.1 PERTUSSIS 1424 BORDETELLA FDTMLGFAI2307 AAA22984.1 PERTUSSIS 1425 BORDETELLA FEGKPALEL 2308 AAA22983.1PERTUSSIS 1426 BORDETELLA FELGADHAV 2309 AGS56996.1 PERTUSSIS 1427BORDETELLA FEPGITTNY 2310 WP_019248658.1 PERTUSSIS 1428 BORDETELLAFETYALTGI 2311 YP_006628018.1 PERTUSSIS 1429 BORDETELLA FIYRETFCI 23121BCP_C PERTUSSIS 1430 BORDETELLA FPTRTTAPG 2313 NP_882284.1 PERTUSSIS1431 BORDETELLA FQTYALTGI 2314 1BCP_C PERTUSSIS 1432 BORDETELLAFTHADGWFL 2315 AGS56996.1 PERTUSSIS 1433 BORDETELLA FVRDGQSVI 23161BCP_C PERTUSSIS 1434 BORDETELLA FVRSGQPVI 2317 YP_006628018.1 PERTUSSIS1435 BORDETELLA FVWYVDTVI 2318 WP_019248866.1 PERTUSSIS 1436 BORDETELLAGAASSRQAL 2319 WP_019249248.1 PERTUSSIS 1437 BORDETELLA GAASSYFEY 2320AAW72734.1 PERTUSSIS 1438 BORDETELLA GAFDLKTTF 2321 AFK26303.1 PERTUSSIS1439 BORDETELLA GAPAAVSVL 2322 AGS56996.1 PERTUSSIS 1440 BORDETELLAGATRAVDSL 2323 AGS56996.1 PERTUSSIS 1441 BORDETELLA GAVPGGAVP 2324AGT50936.1 PERTUSSIS 1442 BORDETELLA GEAMVLVYY 2325 AFK26302.1 PERTUSSIS1443 BORDETELLA GEIALGDAT 2326 WP_019249248.1 PERTUSSIS 1444 BORDETELLAGELMAAQVA 2327 WP_019247158.1 PERTUSSIS 1445 BORDETELLA GGVPGGAVP 2328AAZ74338.1 PERTUSSIS 1446 BORDETELLA GHEHDTWFD 2329 AAA22984.1 PERTUSSIS1447 BORDETELLA GIGALKAGA 2330 WP_019249248.1 PERTUSSIS 1448 BORDETELLAGIVIPPKAL 2331 1BCP_C PERTUSSIS 1449 BORDETELLA GKDLKRPGS 2332AAA22983.1 PERTUSSIS 1450 BORDETELLA GKLPKPVTV 2333 WP_019247158.1PERTUSSIS 1451 BORDETELLA GKSLKKKNQ 2334 WP_019247158.1 PERTUSSIS 1452BORDETELLA GLDVQQGTV 2335 WP_019249248.1 PERTUSSIS 1453 BORDETELLAGLTDGVSRI 2336 WP_019249248.1 PERTUSSIS 1454 BORDETELLA GLYPTYTEW 2337WP_019249248.1 PERTUSSIS 1455 BORDETELLA GLYQTYTEW 2338 YP_006626470.1PERTUSSIS 1456 BORDETELLA GPPSRPTTP 2339 WP_019247158.1 PERTUSSIS 1457BORDETELLA GPSAVAAPA 2340 WP_019249248.1 PERTUSSIS 1458 BORDETELLAGVAPTAQQL 2341 WP_019248866.1 PERTUSSIS 1459 BORDETELLA GVGLASTLW 2342AGS56996.1 PERTUSSIS 1460 BORDETELLA GYEAGFSLG 2343 WP_019247158.1PERTUSSIS 1461 BORDETELLA HADDGTIVI 2344 WP_019248145.1 PERTUSSIS 1462BORDETELLA HADWNNQSI 2345 AGS56996.1 PERTUSSIS 1463 BORDETELLA HAEHEKDVR2346 WP_019247158.1 PERTUSSIS 1464 BORDETELLA HANHYGTRI 2347WP_019247158.1 PERTUSSIS 1465 BORDETELLA HAQGKALLY 2348 AGT50936.1PERTUSSIS 1466 BORDETELLA HFIGYIYEV 2349 AAW72734.1 PERTUSSIS 1467BORDETELLA HLSPALADV 2350 AAA22983.1 PERTUSSIS 1468 BORDETELLA HSLYASYEY2351 AGS56996.1 PERTUSSIS 1469 BORDETELLA HVRGMLVPV 2352 AAA22974.1PERTUSSIS 1470 BORDETELLA HVSKEEQYY 2353 YP_006628018.1 PERTUSSIS 1471BORDETELLA HVTRGWSIF 2354 AFK26303.1 PERTUSSIS 1472 BORDETELLA IADSGFYLD2355 AGS56996.1 PERTUSSIS 1473 BORDETELLA IAHRTELRG 2356 AGS56996.1PERTUSSIS 1474 BORDETELLA IENTAKLSG 2357 WP_019249248.1 PERTUSSIS 1475BORDETELLA IESKISQSV 2358 WP_019249248.1 PERTUSSIS 1476 BORDETELLAIETGGARRF 2359 AGT50936.1 PERTUSSIS 1477 BORDETELLA IIKDAPPGA 23601BCP_C PERTUSSIS 1478 BORDETELLA IIQEFAADL 2361 WP_019249248.1 PERTUSSIS1479 BORDETELLA ILAGALATY 2362 AAW72734.1 PERTUSSIS 1480 BORDETELLAILLENPAAE 2363 AGS56996.1 PERTUSSIS 1481 BORDETELLA ILPILVLAL 2364NP_882286.1 PERTUSSIS 1482 BORDETELLA IPFQRALRL 2365 WP_019248145.1PERTUSSIS 1483 BORDETELLA ISVRVHVSK 2366 YP_006628018.1 PERTUSSIS 1484BORDETELLA ITNETGKTY 2367 WP_019247158.1 PERTUSSIS 1485 BORDETELLAITNKRAALI 2368 WP_019249248.1 PERTUSSIS 1486 BORDETELLA ITSYVGFSV 2369WP_019248658.1 PERTUSSIS 1487 BORDETELLA ITVTSRGGF 2370 WP_019249248.1PERTUSSIS 1488 BORDETELLA IVIPPKALF 2371 1BCP_C PERTUSSIS 1489BORDETELLA IVVEAGELV 2372 WP_019249248.1 PERTUSSIS 1490 BORDETELLAKAAKSVNLM 2373 WP_019247158.1 PERTUSSIS 1491 BORDETELLA KAAPLRRTT 2374AGS56996.1 PERTUSSIS 1492 BORDETELLA KAGKLSATG 2375 WP_019249248.1PERTUSSIS 1493 BORDETELLA KAGTIAAPW 2376 WP_019249248.1 PERTUSSIS 1494BORDETELLA KAKSLTTEI 2377 WP_019249248.1 PERTUSSIS 1495 BORDETELLAKATVTTVQV 2378 WP_019247158.1 PERTUSSIS 1496 BORDETELLA KDYRDKDGG 2379WP_019247158.1 PERTUSSIS 1497 BORDETELLA KEAATIVAA 2380 WP_019249248.1PERTUSSIS 1498 BORDETELLA KEDVDAAQI 2381 WP_019248658.1 PERTUSSIS 1499BORDETELLA KEVDGIIQE 2382 WP_019249248.1 PERTUSSIS 1500 BORDETELLAKGPKLAMPW 2383 AGS56996.1 PERTUSSIS 1501 BORDETELLA KLASGGGAV 2384WP_019249248.1 PERTUSSIS 1502 BORDETELLA KLKGKNQEF 2385 AAA22984.1PERTUSSIS 1503 BORDETELLA KLLHHILPI 2386 NP882286.1 PERTUSSIS 1504BORDETELLA KPAPQPGPQ 2387 AGS56996.1 PERTUSSIS 1505 BORDETELLA KPAPTAPPM2388 WP_019249248.1 PERTUSSIS 1506 BORDETELLA KPAVSVKVA 2389WP_019249248.1 PERTUSSIS 1507 BORDETELLA KPDRAARVA 2390 WP_019249248.1PERTUSSIS 1508 BORDETELLA KPLADIAVI 2391 YP_006626470.1 PERTUSSIS 1509BORDETELLA KPLADIAVV 2392 WP_019249248.1 PERTUSSIS 1510 BORDETELLAKPLPKPLPV 2393 WP_019247158.1 PERTUSSIS 1511 BORDETELLA KQADRDFVW 2394WP_019247699.1 PERTUSSIS 1512 BORDETELLA KSLPGGKLP 2395 WP_019247158.1PERTUSSIS 1513 BORDETELLA KSYTLRYLA 2396 WP_019248658.1 PERTUSSIS 1514BORDETELLA KTNMVVTSV 2397 AAA22983.1 PERTUSSIS 1515 BORDETELLA KVLAPRLYL2398 AAA22974.1 PERTUSSIS 1516 BORDETELLA KVLSTKTTL 2399 WP_019247158.1PERTUSSIS 1517 BORDETELLA LAAGAGLTL 2400 WP_019249248.1 PERTUSSIS 1518BORDETELLA LAAIASAAH 2401 WP_019248145.1 PERTUSSIS 1519 BORDETELLALAANGNGQW 2402 AGS56996.1 PERTUSSIS 1520 BORDETELLA LAAQVTQRG 2403WP_019249248.1 PERTUSSIS 1521 BORDETELLA LAARGDGAL 2404 AAA22974.1PERTUSSIS 1522 BORDETELLA LAAVLVNPH 2405 WP_019249248.1 PERTUSSIS 1523BORDETELLA LAGSGLFRM 2406 AGS56996.1 PERTUSSIS 1524 BORDETELLA LAKALSAAL2407 WP_019249248.1 PERTUSSIS 1525 BORDETELLA LALQAGDAL 2408WP_019249248.1 PERTUSSIS 1526 BORDETELLA LAMPWTFHA 2409 AGS56996.1PERTUSSIS 1527 BORDETELLA LANDGTIVI 2410 WP_019248658.1 PERTUSSIS 1528BORDETELLA LAPTVGVAF 2411 WP_019247158.1 PERTUSSIS 1529 BORDETELLALASDGSVDF 2412 AGS56996.1 PERTUSSIS 1530 BORDETELLA LATYQSEYL 2413AAW72734.1 PERTUSSIS 1531 BORDETELLA LAWALMLAC 2414 AAA22974.1 PERTUSSIS1532 BORDETELLA LEAGRRFTH 2415 AGS56996.1 PERTUSSIS 1533 BORDETELLALFTQQGGAY 2416 1BCPC PERTUSSIS 1534 BORDETELLA LKRPGSSPM 2417 AAA22983.1PERTUSSIS 1535 BORDETELLA LLGSHVARA 2418 AFK26303.1 PERTUSSIS 1536BORDETELLA LLHHILPIL 2419 NP882286.1 PERTUSSIS 1537 BORDETELLA LLNAGGTLL2420 WP_019249248.1 PERTUSSIS 1538 BORDETELLA LMGPLQVNA 2421WP_019249248.1 PERTUSSIS 1539 BORDETELLA LNDSKITMG 2422 WP_019248145.1PERTUSSIS 1540 BORDETELLA LPEPVKLTL 2423 AGT50936.1 PERTUSSIS 1541BORDETELLA LPILVLALL 2424 NP882286.1 PERTUSSIS 1542 BORDETELLA LPKISKNAL2425 WP_019248145.1 PERTUSSIS 1543 BORDETELLA LPKPVTVKL 2426WP_019247158.1 PERTUSSIS 1544 BORDETELLA LPLKANPMH 2427 NP882285.1PERTUSSIS 1545 BORDETELLA LPPRPVVAE 2428 WP_019247158.1 PERTUSSIS 1546BORDETELLA LPSIPGTSI 2429 AGS56996.1 PERTUSSIS 1547 BORDETELLA LPTHLYKNF2430 AAA22984.1 PERTUSSIS 1548 BORDETELLA LPVRGVALV 2431 WP_019249248.1PERTUSSIS 1549 BORDETELLA LPVSLTALD 2432 WP_019249248.1 PERTUSSIS 1550BORDETELLA LQGGNKVPV 2433 WP_019249248.1 PERTUSSIS 1551 BORDETELLALSAALGADW 2434 WP_019249248.1 PERTUSSIS 1552 BORDETELLA LSDAGHEHD 2435AAA22984.1 PERTUSSIS 1553 BORDETELLA LSGEVQRKG 2436 WP_019249248.1PERTUSSIS 1554 BORDETELLA LSSPSAITV 2437 WP_019249248.1 PERTUSSIS 1555BORDETELLA LTWLAILAV 2438 AAW72734.1 PERTUSSIS 1556 BORDETELLA LVFSHVRGM2439 AAA22974.1 PERTUSSIS 1557 BORDETELLA LVSDAGADL 2440 WP_019249248.1PERTUSSIS 1558 BORDETELLA LVYYESIAY 2441 AFK26302.1 PERTUSSIS 1559BORDETELLA MAAESTFES 2442 WP_019247158.1 PERTUSSIS 1560 BORDETELLAMAAGHDATL 2443 WP_019249248.1 PERTUSSIS 1561 BORDETELLA MAAWSERAG 2444AFK26302.1 PERTUSSIS 1562 BORDETELLA MALGALGAA 2445 AGS56996.1 PERTUSSIS1563 BORDETELLA MAPVMGACM 2446 ADA85123.1 PERTUSSIS 1564 BORDETELLAMATKGEMQI 2447 WP_019249248.1 PERTUSSIS 1565 BORDETELLA MDAKGGTLL 2448WP_019249248.1 PERTUSSIS 1566 BORDETELLA MESNKDIVI 2449 WP_019249248.1PERTUSSIS 1567 BORDETELLA MEVMLRAVF 2450 AAA22983.1 PERTUSSIS 1568BORDETELLA MEYFKTPLP 2451 WP_019249248.1 PERTUSSIS 1569 BORDETELLAMHTIASILL 2452 AAA22984.1 PERTUSSIS 1570 BORDETELLA MIYMSGLAV 2453 1BCPCPERTUSSIS 1571 BORDETELLA MLACTGLPL 2454 AAA22974.1 PERTUSSIS 1572BORDETELLA MPIDRKTLC 2455 AFK26303.1 PERTUSSIS 1573 BORDETELLA MPKAPELDL2456 WP_019249248.1 PERTUSSIS 1574 BORDETELLA MQRQAGLPL 2457 NP882285.1PERTUSSIS 1575 BORDETELLA NALAWALML 2458 AAA22974.1 PERTUSSIS 1576BORDETELLA NITNKRAAL 2459 WP_019249248.1 PERTUSSIS 1577 BORDETELLANLMAAESTF 2460 WP_019247158.1 PERTUSSIS 1578 BORDETELLA NNETMSGRQ 2461WP_019249248.1 PERTUSSIS 1579 BORDETELLA NPGSLIAEV 2462 WP_019249248.1PERTUSSIS 1580 BORDETELLA NPMHTIASI 2463 NP882285.1 PERTUSSIS 1581BORDETELLA NPQTQLSNI 2464 WP_019248145.1 PERTUSSIS 1582 BORDETELLANPYTSRRSV 2465 AFK26302.1 PERTUSSIS 1583 BORDETELLA PAPTAPPMP 2466WP_019249248.1 PERTUSSIS 1584 BORDETELLA PASANTLLL 2467 AGS56996.1PERTUSSIS 1585 BORDETELLA PAVALPRPL 2468 WP_019249248.1 PERTUSSIS 1586BORDETELLA PDAHVPFCF 2469 AAA22983.1 PERTUSSIS 1587 BORDETELLA PELGAAIRV2470 AGT50936.1 PERTUSSIS 1588 BORDETELLA PFIIKLKDC 2471 WP_019248145.1PERTUSSIS 1589 BORDETELLA PGPQPPQPP 2472 AGS56996.1 PERTUSSIS 1590BORDETELLA PGPQPPQPQ 2473 AAZ74338.1 PERTUSSIS 1591 BORDETELLA PGPTTDYST2474 WP_019248145.1 PERTUSSIS 1592 BORDETELLA PGTFTAGKD 2475WP_019249248.1 PERTUSSIS 1593 BORDETELLA PGTPGDLLE 2476 AAA22984.1PERTUSSIS 1594 BORDETELLA PKPKPKAER 2477 WP_019247158.1 PERTUSSIS 1595BORDETELLA PKPKPKPKA 2478 WP_019247158.1 PERTUSSIS 1596 BORDETELLAPKPKPKPKP 2479 WP_019247158.1 PERTUSSIS 1597 BORDETELLA PLPPRPVVA 2480WP_019247158.1 PERTUSSIS 1598 BORDETELLA PPAPKPAPQ 2481 AGS56996.1PERTUSSIS 1599 BORDETELLA PPKPAPVAK 2482 WP_019247158.1 PERTUSSIS 1600BORDETELLA PPRPVAAQV 2483 WP_019247158.1 PERTUSSIS 1601 BORDETELLAPPRPVVAEK 2484 WP_019247158.1 PERTUSSIS 1602 BORDETELLA PPSRPTTPP 2485WP_019247158.1 PERTUSSIS 1603 BORDETELLA PRRARRALR 2486 WP_019249248.1PERTUSSIS 1604 BORDETELLA QAAPLSITL 2487 AGT50936.1 PERTUSSIS 1605BORDETELLA QADRDFVWY 2488 WP_019247699.1 PERTUSSIS 1606 BORDETELLAQAIVVGKDL 2489 WP_019249248.1 PERTUSSIS 1607 BORDETELLA QALGALKLY 2490ACI16088.1 PERTUSSIS 1608 BORDETELLA QELALKLKG 2491 AAA22984.1 PERTUSSIS1609 BORDETELLA QITQHGGPY 2492 NP882283.1 PERTUSSIS 1610 BORDETELLAQITQHGSPY 2493 AFK26303.1 PERTUSSIS 1611 BORDETELLA QPLPPRPVA 2494WP_019247158.1 PERTUSSIS 1612 BORDETELLA QPPAGRELS 2495 AGS56996.1PERTUSSIS 1613 BORDETELLA QQLKQADRD 2496 WP_019247699.1 PERTUSSIS 1614BORDETELLA QQVQVLQRQ 2497 WP_019247158.1 PERTUSSIS 1615 BORDETELLAQSIVEAPEL 2498 AGT50936.1 PERTUSSIS 1616 BORDETELLA QVGSSNSAF 2499AAW72734.1 PERTUSSIS 1617 BORDETELLA RAGLSPATW 2500 WP_019249248.1PERTUSSIS 1618 BORDETELLA RARRALRQD 2501 WP_019249248.1 PERTUSSIS 1619BORDETELLA RASASRARI 2502 WP_019249248.1 PERTUSSIS 1620 BORDETELLARELSAAANA 2503 AGS56996.1 PERTUSSIS 1621 BORDETELLA RETFCITTI 2504 1BCPCPERTUSSIS 1622 BORDETELLA RGFAQRQQL 2505 AGS56996.1 PERTUSSIS 1623BORDETELLA RGSAATFTL 2506 WP_019248295.1 PERTUSSIS 1624 BORDETELLARGWSIFALY 2507 AFK26303.1 PERTUSSIS 1625 BORDETELLA RKMLYLIYV 2508YP_006628018.1 PERTUSSIS 1626 BORDETELLA RLRKMLYLI 2509 YP_00662 8018.1PERTUSSIS 1627 BORDETELLA RPQITDAVT 2510 WP_019249248.1 PERTUSSIS 1628BORDETELLA RPSVNGGRI 2511 WP_019249248.1 PERTUSSIS 1629 BORDETELLARRFTHADGW 2512 AGS56996.1 PERTUSSIS 1630 BORDETELLA RSGARATSL 2513AAA22974.1 PERTUSSIS 1631 BORDETELLA RSRVRALAW 2514 NP882284.1 PERTUSSIS1632 BORDETELLA RSRVRALSW 2515 YP_006628019.1 PERTUSSIS 1633 BORDETELLARTHGVGASL 2516 AGS56996.1 PERTUSSIS 1634 BORDETELLA RTRGQARSG 2517AAA22974.1 PERTUSSIS 1635 BORDETELLA RVAPPAVAL 2518 WP_019249248.1PERTUSSIS 1636 BORDETELLA RVLPEPVKL 2519 AGT50936.1 PERTUSSIS 1637BORDETELLA RVRALAWLL 2520 NP882284.1 PERTUSSIS 1638 BORDETELLA RVRALSWLL2521 YP_006628019.1 PERTUSSIS 1639 BORDETELLA RVTVSGGSL 2522 AGT50936.1PERTUSSIS 1640 BORDETELLA SEAMAAWSE 2523 AFK26302.1 PERTUSSIS 1641BORDETELLA SESHNFHAS 2524 WP_019247158.1 PERTUSSIS 1642 BORDETELLASGEGRVNIG 2525 WP_019249248.1 PERTUSSIS 1643 BORDETELLA SGLAVRVHV 25261BCPC PERTUSSIS 1644 BORDETELLA SLADISLGA 2527 WP_019249248.1 PERTUSSIS1645 BORDETELLA SLFAILSST 2528 WP_019247158.1 PERTUSSIS 1646 BORDETELLASLFAPHGNV 2529 AAZ74338.1 PERTUSSIS 1647 BORDETELLA SLSIDNATW 2530AGS56996.1 PERTUSSIS 1648 BORDETELLA SPMEVMLRA 2531 AAA22983.1 PERTUSSIS1649 BORDETELLA SPQPIRATV 2532 WP_019247158.1 PERTUSSIS 1650 BORDETELLASPRRARRAL 2533 WP_019249248.1 PERTUSSIS 1651 BORDETELLA SPSRLAGTL 2534WP_019249248.1 PERTUSSIS 1652 BORDETELLA SSTPLGSLF 2535 WP_019247158.1PERTUSSIS 1653 BORDETELLA STYELLDYL 2536 WP_019249248.1 PERTUSSIS 1654BORDETELLA SVAMKPYEV 2537 AAA22983.1 PERTUSSIS 1655 BORDETELLA SVAPNALAW2538 AAA22974.1 PERTUSSIS 1656 BORDETELLA SVKVAKKLF 2539 WP_019249248.1PERTUSSIS 1657 BORDETELLA TAFMSGRSL 2540 AAA22984.1 PERTUSSIS 1658BORDETELLA TAGATPFDI 2541 WP_019248658.1 PERTUSSIS 1659 BORDETELLATAPVTSPAW 2542 AAW72734.1 PERTUSSIS 1660 BORDETELLA TARTGWLTW 2543AAW72734.1 PERTUSSIS 1661 BORDETELLA TEAQGVQVR 2544 WP_019248145.1PERTUSSIS 1662 BORDETELLA TEVYLEHRM 2545 AAW72734.1 PERTUSSIS 1663BORDETELLA TFEGKPALE 2546 AAA22983.1 PERTUSSIS 1664 BORDETELLA TFTGKVTNG2547 WP_019248658.1 PERTUSSIS 1665 BORDETELLA TGDGGGHTD 2548 AGS56996.1PERTUSSIS 1666 BORDETELLA TLAKALSAA 2549 WP_019249248.1 PERTUSSIS 1667BORDETELLA TLANVGDTW 2550 AGS56996.1 PERTUSSIS 1668 BORDETELLA TLNASNLTL2551 WP_019249248.1 PERTUSSIS 1669 BORDETELLA TLSSAHGNV 2552WP_019249248.1 PERTUSSIS 1670 BORDETELLA TPFDIKLKE 2553 WP_019248658.1PERTUSSIS 1671 BORDETELLA TPFIIKLKD 2554 WP_019248145.1 PERTUSSIS 1672BORDETELLA TPGWSIYGL 2555 1BCPC PERTUSSIS 1673 BORDETELLA TPLGSAATF 2556AGS56996.1 PERTUSSIS 1674 BORDETELLA TPLGSLFAI 2557 WP_019247158.1PERTUSSIS 1675 BORDETELLA TPLPVSLTA 2558 WP_019249248.1 PERTUSSIS 1676BORDETELLA TRQGIMDQY 2559 YP_006626873.1 PERTUSSIS 1677 BORDETELLATSKQDERNY 2560 WP_019247158.1 PERTUSSIS 1678 BORDETELLA TSPYDGKYW 2561YP_006628018.1 PERTUSSIS 1679 BORDETELLA TSRRSVASI 2562 AFK26302.1PERTUSSIS 1680 BORDETELLA TSRTVTMRY 2563 NP879898.1 PERTUSSIS 1681BORDETELLA TTEYPNARY 2564 ADA85123.1 PERTUSSIS 1682 BORDETELLA TTEYSNARY2565 AFK26302.1 PERTUSSIS 1683 BORDETELLA TTLGLEQTF 2566 WP_019247158.1PERTUSSIS 1684 BORDETELLA TVLAAGAGL 2567 WP_019249248.1 PERTUSSIS 1685BORDETELLA TVQELALKL 2568 AAA22984.1 PERTUSSIS 1686 BORDETELLA TVVQRNKHW2569 WP_019247158.1 PERTUSSIS 1687 BORDETELLA VAAAADLAL 2570WP_019249248.1 PERTUSSIS 1688 BORDETELLA VALASQARW 2571 AGS56996.1PERTUSSIS 1689 BORDETELLA VAMKPYEVT 2572 AAA22983.1 PERTUSSIS 1690BORDETELLA VARLVKLQG 2573 WP_019249248.1 PERTUSSIS 1691 BORDETELLAVAVAGGRWH 2574 AGS56996.1 PERTUSSIS 1692 BORDETELLA VEASAITTY 2575WP_019248145.1 PERTUSSIS 1693 BORDETELLA VEDIGGKNY 2576 WP_019247158.1PERTUSSIS 1694 BORDETELLA VEVSSPPPV 2577 WP_019247158.1 PERTUSSIS 1695BORDETELLA VGAGGYEAG 2578 WP_019247158.1 PERTUSSIS 1696 BORDETELLAVGGGEHGRW 2579 WP_019249248.1 PERTUSSIS 1697 BORDETELLA VHVSKEEQY 2580YP_006628018.1 PERTUSSIS 1698 BORDETELLA VIDGQKVLA 2581 AAA22974.1PERTUSSIS 1699 BORDETELLA VIGACTSPY 2582 YP_006628018.1 PERTUSSIS 1700BORDETELLA VKLGGVYEA 2583 AAA22974.1 PERTUSSIS 1701 BORDETELLA VLAPRLYLT2584 AAA22974.1 PERTUSSIS 1702 BORDETELLA VLVKTNMVV 2585 AAA22983.1PERTUSSIS 1703 BORDETELLA VPASGAPAA 2586 AGS56996.1 PERTUSSIS 1704BORDETELLA VPFCFGKDL 2587 AAA22983.1 PERTUSSIS 1705 BORDETELLA VPVSEHCTV2588 AAA22974.1 PERTUSSIS 1706 BORDETELLA VPVTPPKVE 2589 WP_019247158.1PERTUSSIS 1707 BORDETELLA VRTVSAMEY 2590 WP_019249248.1 PERTUSSIS 1708BORDETELLA VSGGSLSAP 2591 AGT50936.1 PERTUSSIS 1709 BORDETELLA VSSATKAKG2592 WP_019248658.1 PERTUSSIS 1710 BORDETELLA VSSPPPVSV 2593WP_019247158.1 PERTUSSIS 1711 BORDETELLA VSVKVAKKL 2594 WP_019249248.1PERTUSSIS 1712 BORDETELLA VTMRYLASY 2595 WP_019248145.1 PERTUSSIS 1713BORDETELLA VTSVAMKPY 2596 AAA22983.1 PERTUSSIS 1714 BORDETELLA VVAEKVTTP2597 WP_019247158.1 PERTUSSIS 1715 BORDETELLA VVDGPPSRP 2598WP_019247158.1 PERTUSSIS 1716 BORDETELLA VVETAQPLP 2599 WP_019247158.1PERTUSSIS 1717 BORDETELLA WLTWLAILA 2600 AAW72734.1 PERTUSSIS 1718BORDETELLA WTFHAGYRY 2601 AGS56996.1 PERTUSSIS 1719 BORDETELLA WVMTDNSNV2602 AGS56996.1 PERTUSSIS 1720 BORDETELLA YAEHGEVSI 2603 WP_019249248.1PERTUSSIS 1721 BORDETELLA YAIDGTACAG 2584 WP_019249248.1 PERTUSSIS 1722BORDETELLA YALKSRIAL 2605 AAA22984.1 PERTUSSIS 1723 BORDETELLA YATNPQTQL2606 WP_019248145.1 PERTUSSIS 1724 BORDETELLA YDTGDLIAY 2607WP_019248658.1 PERTUSSIS 1725 BORDETELLA YEAGFSLGS 2608 WP_019247158.1PERTUSSIS 1726 BORDETELLA YEDATFETY 2609 YP_006628018.1 PERTUSSIS 1727BORDETELLA YENKSSTPL 2610 WP_019247158.1 PERTUSSIS 1728 BORDETELLAYEVTPTRML 2611 AAA22983.1 PERTUSSIS 1729 BORDETELLA YEYIWGLYP 2612WP_019249248.1 PERTUSSIS 1730 BORDETELLA YEYIWGLYQ 2613 YP_006626470.1PERTUSSIS 1731 BORDETELLA YEYSKGPKL 2614 AGS56996.1 PERTUSSIS 1732BORDETELLA YFEPGPTTD 2615 WP_019248145.1 PERTUSSIS 1733 BORDETELLAYIWGLYPTY 2616 WP_019249248.1 PERTUSSIS 1734 BORDETELLA YIWGLYQTY 2617YP_006626470.1 PERTUSSIS 1735 BORDETELLA YLRQITPGW 2618 1BCPC PERTUSSIS1736 BORDETELLA YMIYMSGLA 2619 1BCPC PERTUSSIS 1737 BORDETELLA YPALRAALI2620 WP_019248658.1 PERTUSSIS 1738 BORDETELLA YPGTPGDLL 2621 AAA22984.1PERTUSSIS 1739 BORDETELLA YPTYTEWSV 2622 WP_019249248.1 PERTUSSIS 1740BORDETELLA YQTYTEWSV 2623 YP_006626470.1 PERTUSSIS 1741 BORDETELLAYSTGDLRAY 2624 WP_019248145.1 PERTUSSIS 1742 BORDETELLA YTLRYLASY 2625WP_019248658.1 PERTUSSIS 1743 BORDETELLA YVLVKTNMV 2626 AAA22983.1PERTUSSIS 1744 BORDETELLA YYDYEDATF 2627 YP_006628018.1 PERTUSSIS 1745BORDETELLA AAFIALYPNSQLAPT 2628 Q7VUO5 PERTUSSIS 509 1746 BORDETELLAGGAEYNLALGQRRA 2629 Q7VUO4 PERTUSSIS 509 1747 BORDETELLAGGAEYNLALGQRRADA 2630 Q7VUO4 PERTUSSIS 509 1748 BORDETELLA IALYPNSQLAPT2631 Q7VUO5 PERTUSSIS 509 1749 BORDETELLA KPDQGEVVAVGPGKKTED 2632P0A339.1 PERTUSSIS 509 1750 BORDETELLA KPDQGEVVAVGPGKKTEDG 2633 P0A339.1PERTUSSIS 509 1751 BORDETELLA LAEVLDYHNFVLTQ 2634 Q7VWM1.1 PERTUSSIS 5091752 CORYNEBACTERIUM QSIALSSLMVAQAIP 2635 AAV70486.1 DIPHTHERIAE 1753CORYNEBACTERIUM SIGVLGYQKTVDHTKVNSKLSLF 2636 AAV70486.1 DIPHTHERIAE 1754BORDETELLA AAHADWNNQSIVKT 2637 ABO77783.1 PERTUSSIS 1755 BORDETELLAAALGRG 2638 ABO77783.1 PERTUSSIS 1756 BORDETELLA AALGRGHSLYASYE 2639ABO77783.1 PERTUSSIS 1757 BORDETELLA AAPLRRTTLAMALG 2640 ABO77783.1PERTUSSIS 1758 BORDETELLA AAPLSITLQAGAHA 2641 ABO77783.1 PERTUSSIS 1759BORDETELLA ADAQGDIVATELPS 2642 ABO77783.1 PERTUSSIS 1760 BORDETELLAADSGFYLDATLRAS 2643 ABO77783.1 PERTUSSIS 1761 BORDETELLA AELA 2644ABO77783.1 PERTUSSIS 1762 BORDETELLA AELAVFRAGGGAYR 2645 ABO77783.1PERTUSSIS 1763 BORDETELLA AELQFRNGSVTSSG 2646 ABO77783.1 PERTUSSIS 1764BORDETELLA AGGRWHLGGLAGYT 2647 ABO77783.1 PERTUSSIS 1765 BORDETELLAAGVAAMQGAVVHLQ 2648 ABO77783.1 PERTUSSIS 1766 BORDETELLA AGYTRGDRGFTGDG2649 ABO77783.1 PERTUSSIS 1767 BORDETELLA ALASQARWTGATRA 2650 ABO77783.1PERTUSSIS 1768 BORDETELLA AMPWTFHAGYRYSW 2651 ABO77783.1 PERTUSSIS 1769BORDETELLA AMQGAVVHLQRATIRRGDAP 2652 ABO77783.1 PERTUSSIS 1770BORDETELLA ANGLRVRDE 2653 ABO77783.1 PERTUSSIS 1771 BORDETELLAANGLRVRDEGGSSV 2654 ABO77783.1 PERTUSSIS 1772 BORDETELLA ANKDGKVDIGTYRY2655 ABO77783.1 PERTUSSIS 1773 BORDETELLA APAAVSVLGASELT 2656 ABO77783.1PERTUSSIS 1774 BORDETELLA APPAPKPAPQPGPQ 2657 ABO77783.1 PERTUSSIS 1775BORDETELLA AQGILLENPAAELQ 2658 ABO77783.1 PERTUSSIS 1776 BORDETELLAARWTGATRAVDSLS 2659 ABO77783.1 PERTUSSIS 1777 BORDETELLA ASLEAGRRFTHADG2660 ABO77783.1 PERTUSSIS 1778 BORDETELLA ASYEYSKGPKLAMP 2661 ABO77783.1PERTUSSIS 1779 BORDETELLA ATFTLANKD 2662 ABO77783.1 PERTUSSIS 1780BORDETELLA ATFTLANKDGKVDI 2663 ABO77783.1 PERTUSSIS 1781 BORDETELLAATRAVDSLSIDNAT 2664 ABO77783.1 PERTUSSIS 1782 BORDETELLA DDDGIALYVAGEQAQ2665 ABO77783.1 PERTUSSIS 1783 BORDETELLA DDGIALYVAGEQAQ 2666 ABO77783.1PERTUSSIS 1784 BORDETELLA DGGHITGGRAAGVA 2667 ABO77783.1 PERTUSSIS 1785BORDETELLA DGIRRFLGTVTVKAGK 2668 ABO77783.1 PERTUSSIS 1786 BORDETELLADGSVDFQQPAEAGR 2669 ABO77783.1 PERTUSSIS 1787 BORDETELLA DGYAVKGKYRTHGV2670 ABO77783.1 PERTUSSIS 1788 BORDETELLA DIVATELPSIPGTS 2671 ABO77783.1PERTUSSIS 1789 BORDETELLA DKLVVMQDASGQHR 2672 ABO77783.1 PERTUSSIS 1790BORDETELLA DLGLSDKLVVMQDA 2673 ABO77783.1 PERTUSSIS 1791 BORDETELLADNATWVMTDNSNVGA 2674 ABO77783.1 PERTUSSIS 1792 BORDETELLADNATWVMTDNSNVGALRLA 2675 ABO77783.1 PERTUSSIS 1793 BORDETELLADNRAGRRFDQKVAG 2676 ABO77783.1 PERTUSSIS 1794 BORDETELLA EAGRFKVLTVNTLA2677 ABO77783.1 PERTUSSIS 1795 BORDETELLA ELAQSIVEAPELGA 2678 ABO77783.1PERTUSSIS 1796 BORDETELLA ELGAAIRVGRGARV 2679 ABO77783.1 PERTUSSIS 1797BORDETELLA ELGADHAVAVAGGR 2680 ABO77783.1 PERTUSSIS 1798 BORDETELLAELPSIPGTSIGPLD 2681 ABO77783.1 PERTUSSIS 1799 BORDETELLA EPVKLTLTGGADAQ2682 ABO77783.1 PERTUSSIS 1800 BORDETELLA EQAQASIADSTLQG 2683 ABO77783.1PERTUSSIS 1801 BORDETELLA ERGANVTVQRSAIV 2684 ABO77783.1 PERTUSSIS 1802BORDETELLA ERQHGIHIQGSDPG 2685 ABO77783.1 PERTUSSIS 1803 BORDETELLAEVGKRIELAGGRQV 2686 ABO77783.1 PERTUSSIS 1804 BORDETELLA FDGAGTVHTNGIAH2687 ABO77783.1 PERTUSSIS 1805 BORDETELLA FQQPAEAGRFKVLT 2688 ABO77783.1PERTUSSIS 1806 BORDETELLA FRAGGGAYRAANGL 2689 ABO77783.1 PERTUSSIS 1807BORDETELLA GAHAQGKALLYRVL 2690 ABO77783.1 PERTUSSIS 1808 BORDETELLAGARVTVSGGSLSAP 2691 ABO77783.1 PERTUSSIS 1809 BORDETELLA GAYRAANGLRVRDE2692 ABO77783.1 PERTUSSIS 1810 BORDETELLA GDAPAGGAVPGGAV 2693 ABO77783.1PERTUSSIS 1811 BORDETELLA GGAVPGGAVPGGFG 2694 ABO77783.1 PERTUSSIS 1812BORDETELLA GGAVPGGFGPVLDG 2695 ABO77783.1 PERTUSSIS 1813 BORDETELLAGGFGPVLDGWYGVD 2696 ABO77783.1 PERTUSSIS 1814 BORDETELLA GGLHIGALQSLQPE2697 ABO77783.1 PERTUSSIS 1815 BORDETELLA GGVQIERGANVTVQ 2698 ABO77783.1PERTUSSIS 1816 BORDETELLA GHSLYASYEYSKGP 2699 ABO77783.1 PERTUSSIS 1817BORDETELLA GHTDSVHVGGYATY 2700 ABO77783.1 PERTUSSIS 1818 BORDETELLAGIAHRTELRGTRAE 2701 ABO77783.1 PERTUSSIS 1819 BORDETELLA GKALLYRVLPEPVK2702 ABO77783.1 PERTUSSIS 1820 BORDETELLA GLGMAAALGRGHSL 2703 ABO77783.1PERTUSSIS 1821 BORDETELLA GNVIETGGARRFAP 2704 ABO77783.1 PERTUSSIS 1822BORDETELLA GPLDVALASQARWT 2705 ABO77783.1 PERTUSSIS 1823 BORDETELLAGQHRLWVRN 2706 ABO77783.1 PERTUSSIS 1824 BORDETELLA GRGFAQRQQLDNRA 2707ABO77783.1 PERTUSSIS 1825 BORDETELLA GRLGLEVGKRIELA 2708 ABO77783.1PERTUSSIS 1826 BORDETELLA GRQVQPYIKASVLQ 2709 ABO77783.1 PERTUSSIS 1827BORDETELLA GRRFTHADGWFLEPQAELA 2710 ABO77783.1 PERTUSSIS 1828 BORDETELLAGSEPASANTLLLVQ 2711 ABO77783.1 PERTUSSIS 1829 BORDETELLA GSSVLGRLGLEVGK2712 ABO77783.1 PERTUSSIS 1830 BORDETELLA GTTIKVSGRQAQGI 2713 ABO77783.1PERTUSSIS 1831 BORDETELLA GTVTVKAGKLVADH 2714 ABO77783.1 PERTUSSIS 1832BORDETELLA HAVAVAGGRWHLGG 2715 ABO77783.1 PERTUSSIS 1833 BORDETELLAIELAGGRQVQPYIK 2716 ABO77783.1 PERTUSSIS 1834 BORDETELLA IHIQGSDPGGVRTA2717 ABO77783.1 PERTUSSIS 1835 BORDETELLA IRRFLGTVTVKAGK 2718 ABO77783.1PERTUSSIS 1836 BORDETELLA IRVGRGARVTVSGG 2719 ABO77783.1 PERTUSSIS 1837BORDETELLA ITLQAGAHA 2720 ABO77783.1 PERTUSSIS 1838 BORDETELLAITLQAGAHAQGKAL 2721 ABO77783.1 PERTUSSIS 1839 BORDETELLA IVEAPELGAAIRVG2722 ABO77783.1 PERTUSSIS 1840 BORDETELLA IVKTGERQHGIHIQ 2723 ABO77783.1PERTUSSIS 1841 BORDETELLA KAGKLVADHATLAN 2724 ABO77783.1 PERTUSSIS 1842BORDETELLA KGKYRTHGVGASLE 2725 ABO77783.1 PERTUSSIS 1843 BORDETELLAKPAPQPGPQPPQPP 2726 ABO77783.1 PERTUSSIS 1844 BORDETELLA KVAGFELGADHAVA2727 ABO77783.1 PERTUSSIS 1845 BORDETELLA KVAGSDGYAVKGKY 2728 ABO77783.1PERTUSSIS 1846 BORDETELLA KVDIGTYRYRLAAN 2729 ABO77783.1 PERTUSSIS 1847BORDETELLA KVLTVNTLAGSGLF 2730 ABO77783.1 PERTUSSIS 1848 BORDETELLALAANGNGQWSLVGA 2731 ABO77783.1 PERTUSSIS 1849 BORDETELLA LAMPWTFHAGYRYS2732 ABO77783.1 PERTUSSIS 1850 BORDETELLA LASTLWYAESNALS 2733 ABO77783.1PERTUSSIS 1851 BORDETELLA LENDFKVAGSDGYA 2734 ABO77783.1 PERTUSSIS 1852BORDETELLA LENPAAELQFRNGS 2735 ABO77783.1 PERTUSSIS 1853 BORDETELLALGAAPAAHADWNNQ 2736 ABO77783.1 PERTUSSIS 1854 BORDETELLALGGLAGYTRGDRGFTGDG 2737 ABO77783.1 PERTUSSIS 1855 BORDETELLA LLENP 2738ABO77783.1 PERTUSSIS 1856 BORDETELLA LLVQTPLGSAATFT 2739 ABO77783.1PERTUSSIS 1857 BORDETELLA LPPSRVVLRDTNVT 2740 ABO77783.1 PERTUSSIS 1858BORDETELLA LQPEDLPPS 2741 ABO77783.1 PERTUSSIS 1859 BORDETELLALQPEDLPPSRVVLR 2742 ABO77783.1 PERTUSSIS 1860 BORDETELLA LRASRLENDFKVAG2743 ABO77783.1 PERTUSSIS 1861 BORDETELLA LRLASDGSVDFQQP 2744 ABO77783.1PERTUSSIS 1862 BORDETELLA LSAAANAAVNTGGV 2745 ABO77783.1 PERTUSSIS 1863BORDETELLA LSAPHGNVIETGGA 2746 ABO77783.1 PERTUSSIS 1864 BORDETELLALSDDGIRRFLGTVT 2747 ABO77783.1 PERTUSSIS 1865 BORDETELLA LVGAKAPPAPKPAP2748 ABO77783.1 PERTUSSIS 1866 BORDETELLA LYVAGEQAQASIAD 2749 ABO77783.1PERTUSSIS 1867 BORDETELLA MALGALGAAPAAHA 2750 ABO77783.1 PERTUSSIS 1868BORDETELLA MNMSLSRIVKAAPL 2751 ABO77783.1 PERTUSSIS 1869 BORDETELLAMQDASGQHR 2752 ABO77783.1 PERTUSSIS 1870 BORDETELLA MQGAVVHLQRATIR 2753ABO77783.1 PERTUSSIS 1871 BORDETELLA NAAVNTGGVGLAST 2754 ABO77783.1PERTUSSIS 1872 BORDETELLA NALSKRLGELRLNP 2755 ABO77783.1 PERTUSSIS 1873BORDETELLA NGQWSLVGAKAPPA 2756 ABO77783.1 PERTUSSIS 1874 BORDETELLANTLAGSGLFRMNVF 2757 ABO77783.1 PERTUSSIS 1875 BORDETELLA PAGRELSAAANAAV2758 ABO77783.1 PERTUSSIS 1876 BORDETELLA PAPQPPAGRELSAA 2759 ABO77783.1PERTUSSIS 1877 BORDETELLA PGPQPPQPPQPQPE 2760 ABO77783.1 PERTUSSIS 1878BORDETELLA PGTSIGPLDVALAS 2761 ABO77783.1 PERTUSSIS 1879 BORDETELLAPLGSAATFTLANKD 2762 ABO77783.1 PERTUSSIS 1880 BORDETELLA PQPEAPAPQPPAGR2763 ABO77783.1 PERTUSSIS 1881 BORDETELLA PQPPQPQPEAPAPQ 2764 ABO77783.1PERTUSSIS 1882 BORDETELLA PYIKASVLQEFDGA 2765 ABO77783.1 PERTUSSIS 1883BORDETELLA RFAPQAAPLSITLQ 2766 ABO77783.1 PERTUSSIS 1884 BORDETELLARLGELRLNPDAGGA 2767 ABO77783.1 PERTUSSIS 1885 BORDETELLA RLNPDAGGAWGRGF2768 ABO77783.1 PERTUSSIS 1886 BORDETELLA RNGSVTSSGQLSDD 2769 ABO77783.1PERTUSSIS 1887 BORDETELLA RRFDQKVAGFELGA 2770 ABO77783.1 PERTUSSIS 1888BORDETELLA RTTLAMALGALGAA 2771 ABO77783.1 PERTUSSIS 1889 BORDETELLASAIVDGGLHIGALQ 2772 ABO77783.1 PERTUSSIS 1890 BORDETELLA SANTLLLVQTPLGS2773 ABO77783.1 PERTUSSIS 1891 BORDETELLA SDPGGVRTASGTTI 2774 ABO77783.1PERTUSSIS 1892 BORDETELLA SELTLDGGHITGGR 2775 ABO77783.1 PERTUSSIS 1893BORDETELLA SGLFRMNVF 2776 ABO77783.1 PERTUSSIS 1894 BORDETELLASGLFRMNVFADLGL 2777 ABO77783.1 PERTUSSIS 1895 BORDETELLA SGSSVELAQSIVEA2778 ABO77783.1 PERTUSSIS 1896 BORDETELLA SIADSTLQGAGGVQ 2779 ABO77783.1PERTUSSIS 1897 BORDETELLA SKGPKLAMPWTFHA 2780 ABO77783.1 PERTUSSIS 1898BORDETELLA SNVGALRLASDGSV 2781 ABO77783.1 PERTUSSIS 1899 BORDETELLASRIVKAAPLRRTTL 2782 ABO77783.1 PERTUSSIS 1900 BORDETELLA SVLGASELTLDGGH2783 ABO77783.1 PERTUSSIS 1901 BORDETELLA SVLQEFDGA 2784 ABO77783.1PERTUSSIS 1902 BORDETELLA SVLQEFDGAGTVHT 2785 ABO77783.1 PERTUSSIS 1903BORDETELLA TELR 2786 ABO77783.1 PERTUSSIS 1904 BORDETELLA TELRGTRAELGLGM2787 ABO77783.1 PERTUSSIS 1905 BORDETELLA TGDGGGHTDSVHVG 2788 ABO77783.1PERTUSSIS 1906 BORDETELLA TGGARRFAPQAAPL 2789 ABO77783.1 PERTUSSIS 1907BORDETELLA TGGRAAGVAAMQGA 2790 ABO77783.1 PERTUSSIS 1908 BORDETELLATGGVGLASTLWYAE 2791 ABO77783.1 PERTUSSIS 1990 BORDETELLA THGVGASLEAGRRF2792 ABO77783.1 PERTUSSIS 1910 BORDETELLA TIRRGDAPA 2793 ABO77783.1PERTUSSIS 1911 BORDETELLA TLANVGDTWDDDGI 2794 ABO77783.1 PERTUSSIS 1912BORDETELLA TLQGAGGVQIERGA 2795 ABO77783.1 PERTUSSIS 1913 BORDETELLATLTGGADAQGDIVA 2796 ABO77783.1 PERTUSSIS 1914 BORDETELLA TNVTAVPASGAPAA2797 ABO77783.1 PERTUSSIS 1915 BORDETELLA TRAELGLGMAAALG 2798 ABO77783.1PERTUSSIS 1916 BORDETELLA TSSGQLSDDGIRRF 2799 ABO77783.1 PERTUSSIS 1917BORDETELLA TVHTNGIAHRTELR 2800 ABO77783.1 PERTUSSIS 1918 BORDETELLATYRYRLAANGNGQW 2801 ABO77783.1 PERTUSSIS 1919 BORDETELLA VADHATLANVGDTW2802 ABO77783.1 PERTUSSIS 1920 BORDETELLA VHVGGYATYIADSG 2803 ABO77783.1PERTUSSIS 1921 BORDETELLA VLDGWYGVD 2804 ABO77783.1 PERTUSSIS 1922BORDETELLA VPASGAPAAVSVLG 2805 ABO77783.1 PERTUSSIS 1923 BORDETELLAVRDEGGSSVLGRLG 2806 ABO77783.1 PERTUSSIS 1924 BORDETELLA VRTASGTTIKVSGR2807 ABO77783.1 PERTUSSIS 1925 BORDETELLA VSGGSLSAPHGNVI 2808 ABO77783.1PERTUSSIS 1926 BORDETELLA VSGRQAQGILLENP 2809 ABO77783.1 PERTUSSIS 1927BORDETELLA VTVQRSAIVDGGLH 2810 ABO77783.1 PERTUSSIS 1928 BORDETELLAVVLRDTNVTAVPAS 2811 ABO77783.1 PERTUSSIS 1929 BORDETELLA WNNQSIVKTGERQH2812 ABO77783.1 PERTUSSIS 1930 BORDETELLA WVRNSGSEPASANT 2813 ABO77783.1PERTUSSIS 1931 BORDETELLA WYAESNALSKRLGE 2814 ABO77783.1 PERTUSSIS 1932BORDETELLA YATYIADSGFYLDA 2815 ABO77783.1 PERTUSSIS 1933 BORDETELLAYGVDVSGSS 2816 ABO77783.1 PERTUSSIS 1934 BORDETELLA YGVDVSGSSVELAQ 2817ABO77783.1 PERTUSSIS 1935 BORDETELLA YLDATLRASRLEND 2818 ABO77783.1PERTUSSIS 1936 BORDETELLA YRVLPEPVKLTLTG 2819 ABO77783.1 PERTUSSIS 1937BORDETELLA VKAQNITNKRAALIEA 2820 AAA22974.1 PERTUSSIS 1938 BORDETELLAYYSNVTATRLLSSTNS 2821 AAA83981.1 PERTUSSIS 1939 BORDETELLASPNLTDERAAQAGVT 2822 CPP72976.1 PERTUSSIS 1940 MEASLES SSRASDERAAHLPTS2823 BAA33867.1 MORBILLIVIRUS 1941 CORYNEBACTERIUM QVVHNSYNRPAYSPG 28241007216A DIPHTHERIAE 1942 MEASLES VIRUS AEGGEIHEL 2825 AAF85692.1 STRAINEDMONSTON-B 1943 MEASLES VIRUS AENLISNGIGKY 2826 AAF85698.1 STRAINEDMONSTON-B 1944 MEASLES VIRUS AEVDGDVKL 2827 CAB43772.1 STRAINEDMONSTON-B 1945 MEASLES VIRUS AIYTAEIHK 2828 AAF85697.1 STRAINEDMONSTON-B 1946 MEASLES VIRUS APVFHMTNY 2829 CAB43772.1 STRAINEDMONSTON-B 1947 MEASLES VIRUS APVFHMTNYLEQPVSN 2830 AAR89413.1 STRAINEDMONSTON-B 1948 MEASLES VIRUS AQRLNEIY 2831 AAF85698.1 STRAINEDMONSTON-B 1949 MEASLES VIRUS ARVPHAYSL 2832 AAF85698.1 STRAINEDMONSTON-B 1950 MEASLES VIRUS AVRDLERAM 2833 P03424.1 STRAINEDMONSTON-B 1951 MEASLES VIRUS AVRDLERAMTTLK 2834 P03424.1 STRAINEDMONSTON-B 1952 MEASLES VIRUS DALLRLQAM 2835 Q89933.1 STRAINEDMONSTON-B 1953 MEASLES VIRUS DIKEKVINL 2836 AAF85698.1 STRAINEDMONSTON-B 1954 MEASLES VIRUS DQGLFKVL 2837 AAF85695.1 STRAINEDMONSTON-B 1955 MEASLES VIRUS DTGVDTRIW 2838 Q9EMA9.1 STRAINEDMONSTON-B 1956 MEASLES VIRUS EPIGSLAIEEAM 2839 AAF85692.1 STRAINEDMONSTON-B 1957 MEASLES VIRUS EPIRDALNAM 2840 P69354.1 STRAINEDMONSTON-B 1958 MEASLES VIRUS FPKLGKTL 2841 AAF85692.1 STRAINEDMONSTON-B 1959 MEASLES VIRUS FRSVNAVAF 2842 AAF85695.1 STRAINEDMONSTON-B 1960 MEASLES VIRUS GKIIDNTEQL 2843 AAF85695.1 STRAINEDMONSTON-B 1961 MEASLES VIRUS GLNEKLVFY 2844 AAF85695.1 STRAINEDMONSTON-B 1962 MEASLES VIRUS GMYGGTYLVEK 2845 AAC35876.2 STRAINEDMONSTON-B 1963 MEASLES VIRUS GPPISLERLDVGTN 2846 P69354.1 STRAINEDMONSTON-B 1964 MEASLES VIRUS GPRQAQVSFL 2847 Q89933.1 STRAINEDMONSTON-B 1965 MEASLES VIRUS GRLVPQVRVID 2848 AAF85695.1 STRAINEDMONSTON-B 1966 MEASLES VIRUS GSAPISMGFR 2849 AAF85692.1 STRAINEDMONSTON-B 1967 MEASLES VIRUS HILAKSTAL 2850 AAF85698.1 STRAINEDMONSTON-B 1968 MEASLES VIRUS HYREVNLVY 2851 AAF85698.1 STRAINEDMONSTON-B 1969 MEASLES VIRUS IPPMKNLAL 2852 AAC35876.2 STRAINEDMONSTON-B 1970 MEASLES VIRUS IPYQGSGKGVSF 2853 CAB43772.1 STRAINEDMONSTON-B 1971 MEASLES VIRUS ISKESQHVY 2854 AAF85698.1 STRAINEDMONSTON-B 1972 MEASLES VIRUS IVSSHFFVY 2855 AAF85698.1 STRAINEDMONSTON-B 1973 MEASLES VIRUS KEIKETGRLF 2856 AAF85698.1 STRAINEDMONSTON-B 1974 MEASLES VIRUS KESQHVYYL 2857 AAF85698.1 STRAINEDMONSTON-B 1975 MEASLES VIRUS KIIDNTEQL 2858 AAF85695.1 STRAINEDMONSTON-B 1976 MEASLES VIRUS KKQINRQN 2859 AAA63285.1 STRAINEDMONSTON-B 1977 MEASLES VIRUS KKVDTNFIYQ 2860 AAF85698.1 STRAINEDMONSTON-B 1978 MEASLES VIRUS KLIDGFFPA 2861 AAF85698.1 STRAINEDMONSTON-B 1979 MEASLES VIRUS KPNLSSKRSEL 2862 BAB39848.1 STRAINEDMONSTON-B 1980 MEASLES VIRUS KVSPYLFTV 2863 AAR89413.1 STRAINEDMONSTON-B 1981 MEASLES VIRUS LETRTTNQFL 2864 CAB43772.1 STRAINEDMONSTON-B 1982 MEASLES VIRUS LLKEATEL 2865 AAF85695.1 STRAINEDMONSTON-B 1983 MEASLES VIRUS LLKKGNSLY 2866 AAF85698.1 STRAINEDMONSTON-B 1984 MEASLES VIRUS LPAPIGGMNY 2867 AAF85698.1 STRAINEDMONSTON-B 1985 MEASLES VIRUS MPEETLHQVM 2868 AAF85698.1 STRAINEDMONSTON-B 1986 MEASLES VIRUS PTTIRGQFS 2869 CAB43772.1 STRAINEDMONSTON-B 1987 MEASLES VIRUS QEISRHQALGY 2870 P03424.1 STRAINEDMONSTON-B 1988 MEASLES VIRUS RITHVDTESY 2871 P69354.1 STRAINEDMONSTON-B 1989 MEASLES VIRUS RPGLKPDL 2872 P69354.1 STRAIN EDMONSTON-B1990 MEASLES VIRUS RPIYGLEV 2873 AAF85698.1 STRAIN EDMONSTON-B 1991MEASLES VIRUS RQAGQEMILAV 2874 P69354.1 STRAIN EDMONSTON-B 1992MEASLES VIRUS SAVRIATVY 2875 AAF85698.1 STRAIN EDMONSTON-B 1993MEASLES VIRUS SLMPEETLHQV 2876 AAF85698.1 STRAIN EDMONSTON-B 1994MEASLES VIRUS SMIDLVTKF 2877 AAF85698.1 STRAIN EDMONSTON-B 1995MEASLES VIRUS SMLNSQAIDNLRA 2878 P69354.1 STRAIN EDMONSTON-B 1996MEASLES VIRUS SMYRVFEV 2879 CAB43772.1 STRAIN EDMONSTON-B 1997MEASLES VIRUS SQQGMFHAY 2880 AAF85698.1 STRAIN EDMONSTON-B 1998MEASLES VIRUS TDTPIVYNDRNLLD 2881 Q89933.1 STRAIN EDMONSTON-B 1999MEASLES VIRUS VIINDDQGLFKV 2882 AAF85695.1 STRAIN EDMONSTON-B 2000MEASLES VIRUS YESGVRIASL 2883 AAF85698.1 STRAIN EDMONSTON-B 2001MEASLES VIRUS YLKDKALA 2884 AAF85698.1 STRAIN EDMONSTON-B 2002MEASLES VIRUS YVYDHSGEAVK 2885 AAF85692.1 STRAIN EDMONSTON-B 2003RUBELLA VIRUS ARVIDPAAQSFTGVV 2886 BAA28178.1 2004 RUBELLA VIRUSSDRASARVIDPAAQS 2887 BAA28178.1 2005 RUBELLA VIRUS VPPGKFVTAALLNTP 2888BAA28178.1 2006 RUBELLA VIRUS WVTPVIGSQARKCGL 2889 BAA28178.1 2007 MUMPSGTYRLIPNARANLTA 400 AGC97176.1 RUBULAVIRUS

I. Delivery of Prime Editors

In another aspect, the present disclosure provides for the delivery ofprime editors in vitro and in vivo using various strategies, includingon separate vectors using split inteins and as well as direct deliverystrategies of the ribonucleoprotein complex (i.e., the prime editorcomplexed to the PEgRNA and/or the second-site gRNA) using techniquessuch as electroporation, use of cationic lipid-mediated formulations,and induced endocytosis methods using receptor ligands fused to theribonucleoprotein complexes. Any such methods are contemplated herein.

Overview of Delivery Options

In some aspects, the invention provides methods comprising deliveringone or more prime editor-encoding polynucleotides, such as or one ormore vectors as described herein encoding one or more components of theprime editing system described herein, one or more transcripts thereof,and/or one or proteins transcribed therefrom, to a host cell. In someaspects, the invention further provides cells produced by such methods,and organisms (such as animals, plants, or fungi) comprising or producedfrom such cells. In some embodiments, a prime editor as described hereinin combination with (and optionally complexed with) a guide sequence isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a prime editor to cells in culture, or in ahost organism. Non-viral vector delivery systems include DNA plasmids,RNA (e.g. a transcript of a vector described herein), naked nucleicacid, and nucleic acid complexed with a delivery vehicle, such as aliposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. For a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Bihm (eds) (1995); and Yu et al., GeneTherapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386,4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a viruses can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and ψ2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In various embodiments, the PE constructs (including, thesplit-constructs) may be engineered for delivery in one or more rAAVvectors. An rAAV as related to any of the methods and compositionsprovided herein may be of any serotype including any derivative orpseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2/1, 2/5,2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). An rAAV may comprise a genetic load(i.e., a recombinant nucleic acid vector that expresses a gene ofinterest, such as a whole or split PE fusion protein that is carried bythe rAAV into a cell) that is to be delivered to a cell. An rAAV may bechimeric.

As used herein, the serotype of an rAAV refers to the serotype of thecapsid proteins of the recombinant virus. Non-limiting examples ofderivatives and pseudotypes include rAAV2/1, rAAV2/5, rAAV2/8, rAAV2/9,AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15,AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8,AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2,AAV clone 32/83, AAVShH10, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4,AAVM41, and AAVr3.45. A non-limiting example of derivatives andpseudotypes that have chimeric VP1 proteins is rAAV2/5-1VP1u, which hasthe genome of AAV2, capsid backbone of AAV5 and VP1u of AAV1. Othernon-limiting example of derivatives and pseudotypes that have chimericVP1 proteins are rAAV2/5-8VP1u, rAAV2/9-1VP1u, and rAAV2/9-8VP1u.

AAV derivatives/pseudotypes, and methods of producing suchderivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012April; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan. 24. TheAAV vector toolkit: poised at the clinical crossroads. Asokan A1,Schaffer D V, Samulski R J.). Methods for producing and usingpseudotyped rAAV vectors are known in the art (see, e.g., Duan et al.,J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532,2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio etal., Hum. Molec. Genet., 10:3075-3081, 2001).

Methods of making or packaging rAAV particles are known in the art andreagents are commercially available (see, e.g., Zolotukhin et al.Production and purification of serotype 1, 2, and 5 recombinantadeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S.Patent Publication Numbers US20070015238 and US20120322861, which areincorporated herein by reference; and plasmids and kits available fromATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a geneof interest may be combined with one or more helper plasmids, e.g., thatcontain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and acap gene (encoding VP1, VP2, and VP3, including a modified VP2 region asdescribed herein), and transfected into a recombinant cells such thatthe rAAV particle can be packaged and subsequently purified.

Recombinant AAV may comprise a nucleic acid vector, which may compriseat a minimum: (a) one or more heterologous nucleic acid regionscomprising a sequence encoding a protein or polypeptide of interest oran RNA of interest (e.g., a siRNA or microRNA), and (b) one or moreregions comprising inverted terminal repeat (ITR) sequences (e.g.,wild-type ITR sequences or engineered ITR sequences) flanking the one ormore nucleic acid regions (e.g., heterologous nucleic acid regions).Herein, heterologous nucleic acid regions comprising a sequence encodinga protein of interest or RNA of interest are referred to as genes ofinterest.

Any one of the rAAV particles provided herein may have capsid proteinsthat have amino acids of different serotypes outside of the VP1u region.In some embodiments, the serotype of the backbone of the VP1 protein isdifferent from the serotype of the ITRs and/or the Rep gene. In someembodiments, the serotype of the backbone of the VP1 capsid protein of aparticle is the same as the serotype of the ITRs. In some embodiments,the serotype of the backbone of the VP1 capsid protein of a particle isthe same as the serotype of the Rep gene. In some embodiments, capsidproteins of rAAV particles comprise amino acid mutations that result inimproved transduction efficiency.

In some embodiments, the nucleic acid vector comprises one or moreregions comprising a sequence that facilitates expression of the nucleicacid (e.g., the heterologous nucleic acid), e.g., expression controlsequences operatively linked to the nucleic acid. Numerous suchsequences are known in the art. Non-limiting examples of expressioncontrol sequences include promoters, insulators, silencers, responseelements, introns, enhancers, initiation sites, termination signals, andpoly(A) tails. Any combination of such control sequences is contemplatedherein (e.g., a promoter and an enhancer).

Final AAV constructs may incorporate a sequence encoding the PEgRNA. Inother embodiments, the AAV constructs may incorporate a sequenceencoding the second-site nicking guide RNA. In still other embodiments,the AAV constructs may incorporate a sequence encoding the second-sitenicking guide RNA and a sequence encoding the PEgRNA.

In various embodiments, the PEgRNAs and the second-site nicking guideRNAs can be expressed from an appropriate promoter, such as a human U6(hU6) promoter, a mouse U6 (mU6) promoter, or other appropriatepromoter. The PEgRNAs and the second-site nicking guide RNAs can bedriven by the same promoters or different promoters.

In some embodiments, a rAAV constructs or the herein compositions areadministered to a subject enterally. In some embodiments, a rAAVconstructs or the herein compositions are administered to the subjectparenterally. In some embodiments, a rAAV particle or the hereincompositions are administered to a subject subcutaneously,intraocularly, intravitreally, subretinally, intravenously (IV),intracerebro-ventricularly, intramuscularly, intrathecally (IT),intracisternally, intraperitoneally, via inhalation, topically, or bydirect injection to one or more cells, tissues, or organs. In someembodiments, a rAAV particle or the herein compositions are administeredto the subject by injection into the hepatic artery or portal vein.

Split PE Vector-Based Strategies

In this aspect, the prime editors can be divided at a split site andprovided as two halves of a whole/complete prime editor. The two halvescan be delivered to cells (e.g., as expressed proteins or on separateexpression vectors) and once in contact inside the cell, the two halvesform the complete prime editor through the self-splicing action of theinteins on each prime editor half. Split intein sequences can beengineered into each of the halves of the encoded prime editor tofacilitate their transplicing inside the cell and the concomitantrestoration of the complete, functioning PE.

These split intein-based methods overcome several barriers to in vivodelivery. For example, the DNA encoding prime editors is larger than therAAV packaging limit, and so requires special solutions. One suchsolution is formulating the editor fused to split intein pairs that arepackaged into two separate rAAV particles that, when co-delivered to acell, reconstitute the functional editor protein. Several other specialconsiderations to account for the unique features of prime editing aredescribed, including the optimization of second-site nicking targets andproperly packaging prime editors into virus vectors, includinglentiviruses and rAAV.

In this aspect, the prime editors can be divided at a split site andprovided as two halves of a whole/complete prime editor. The two halvescan be delivered to cells (e.g., as expressed proteins or on separateexpression vectors) and once in contact inside the cell, the two halvesform the complete prime editor through the self-splicing action of theinteins on each prime editor half. Split intein sequences can beengineered into each of the halves of the encoded prime editor tofacilitate their transplicing inside the cell and the concomitantrestoration of the complete, functioning PE.

FIG. 66 depicts one embodiment of a prime editor being provided as twoPE half proteins which regenerate as whole prime editor through theself-splicing action of the split-intein halves located at the end orbeginning of each of the prime editor half proteins. As used herein, theterm “PE N-terminal half” refers to the N-terminal half of a completeprime editor and which comprises the “N intein” at the C-terminal end ofthe PE N-terminal half (i.e., the N-terminal extein) of the completeprime editor. The “N intein” refers to the N-terminal half of acomplete, fully-formed split-intein moiety. As used herein, the term “PEC-terminal half” refers to the C-terminal half of a complete primeeditor and which comprises the “C intein” at the N-terminal end of theC-terminal half (i.e., the C-terminal extein) of a complete primeeditor. When the two half proteins, i.e., the PE N-terminal half and thePE C-terminal half, come into contact with one another, e.g., within thecell, the N intein and the C intein undergo the simultaneous process ofself-excision and the formation of a peptide bond between the C-terminalend of the PE N-terminal half and the N-terminal end of the PEC-terminal half to reform the complete prime editor protein comprisingthe complete napDNAbp domain (e.g., Cas9 nickase) and the RT domain.Although not shown in the drawing, the prime editor may also compriseadditional sequences including NLS at the N-terminus and/or C-terminus,as well as amino acid linkers sequences joining each domain.

In various embodiments, the prime editors may be engineered as two halfproteins (i.e., a PE N-terminal half and a PE C-terminal half) by“splitting” the whole prime editor as a “split site.” The “split site”refers to the location of insertion of split intein sequences (i.e., theN intein and the C intein) between two adjacent amino acid residues inthe prime editor. More specifically, the “split site” refers to thelocation of dividing the whole prime editor into two separate halves,wherein in each halve is fused at the split site to either the N inteinor the C intein motifs. The split site can be at any suitable locationin the prime editor fusion protein, but preferably the split site islocated at a position that allows for the formation of two half proteinswhich are appropriately sized for delivery (e.g., by expression vector)and wherein the inteins, which are fused to each half protein at thesplit site termini, are available to sufficiently interact with oneanother when one half protein contacts the other half protein inside thecell.

In some embodiments, the split site is located in the napDNAbp domain.In other embodiments, the split site is located in the RT domain. Inother embodiments, the split site is located in a linker that joins thenapDNAbp domain and the RT domain.

In various embodiments, split site design requires finding sites tosplit and insert an N- and C-terminal intein that are both structurallypermissive for purposes of packaging the two half prime editor domainsinto two different AAV genomes. Additionally, intein residues necessaryfor trans splicing can be incorporated by mutating residues at the Nterminus of the C terminal extein or inserting residues that will leavean intein “scar.”

Exemplary split configurations of split prime editors comprising eitherthe SpCas9 nickase or the SaCas9 nickase are as follows.

S. PYOGENES PE, SPLIT AT LINKER, N TERMINAL PORTIONSTRUCTURE:[N EXTEIN]−[N INTEIN] (SEQ ID NO: 443) MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGG D SGGSSGGSCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNSGGSKRTADGSEFEPKKKRKV KEY: NLS (SEQ ID NO: 124, 125) CAS9 (SEQ ID NO: 445)LINKER  (SEQ ID NO: 446) NPUN INTEIN (SEQ ID NO: 447)S. PYOGENES PE, SPLIT AT LINKER, C TERMINAL PORTIONSTRUCTURE: [C INTEIN]−[C EXTEIN] (SEQ ID NO: 450) MKRTADGSEFESPKKKRKVIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN CFNS GSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLVPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSKRTADGSEFEPKKKRKV KEY: NLS (SEQ ID NO: 124, 125) LINKER 1  (SEQ ID NO: 453)LINKER 2  (SEQ ID NO: 174) NPUC INTEIN (SEQ ID NO: 452) RT (SEQ ID NO: 454)S. AUREUS PE, SPLIT BETWEEN RESIDUES 740/741, N TERMINAL PORTIONSTRUCTURE: [N EXTEIN]−[N INTEIN] (SEQ ID NO: 458) MKRTADGSEFESPKKKRKVGKRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAECLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN SGGSKRTADGSEFEPKKKRKV KEY: NLS (SEQ ID NO: 124, 125) CAS9 (SEQ ID NO: 460)LINKER  (SEQ ID NO: 174) NPUN INTEIN (SEQ ID NO: 462)S. AUREUS PE, SPLIT BETWEEN RESIDUES 740/741, C TERMINAL PORTIONSTRUCTURE: [C INTEIN]−[C EXTEIN] (SEQ ID NO: 465) MKRTADGSEFESPKKKRKVIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN CFNEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG SGGSSGGSSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDIADFRIQHPDLILLQYVDDLLIAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLWEGORKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSKRTADGSEFEPKKKRKV KEY: NLS (SEQ ID NO: 124, 125) CAS9 (SEQ ID NO: 467)LINKER 1  (SEQ ID NO: 127) LINKER 2  (SEQ ID NO: 174)NPUC INTEIN (SEQ ID NO: 452) RT  (SEQ ID NO: 471)

In various embodiments, using SpCas9 nickase (SEQ ID NO: 18, 1368 aminoacids) as an example, the split can between any two amino acids between1 and 1368. Preferred splits, however, will be located between thecentral region of the protein, e.g., from amino acids 50-1250, or from100-1200, or from 150-1150, or from 200-1100, or from 250-1050, or from300-1000, or from 350-950, or from 400-900, or from 450-850, or from500-800, or from 550-750, or from 600-700 of SEQ ID NO: 18. In specificexemplary embodiments, the split site may be between 740741, or 801/802,or 1010/1011, or 1041/1042. In other embodiments the split site may bebetween 1/2, 2/3, 3/4, 4/5, 5/6, 6/7, 7/8, 8/9, 9/10, 10/11, 12/13,14/15, 15/16, 17/18, 19/20, 20/21, 21/22, 22/23, 23/24, 24/25, 25/26,26/27, 27/28, 28/29, 29/30, 30/31, 31/32, 32/33, 33/34, 34/35, 35/36,36/37, 38/39, 39/40, 41/42, 42/43, 43/44, 44/45, 45/46, 46/47, 47/48,48/49, 49/50, 51/52, 52/53, 53/54, 54/55, 55/56, 56/57, 57/58, 58/59,59/60, 61/62, 62/63, 63/64, 64/65, 65/66, 66/67, 67/68, 68/69, 69/70,71/72, 72/73, 73/74, 74/75, 75/76, 76/77, 77/78, 78/79, 79/80, 81/82,82/83, 83/84, 84/85, 85/86, 86/87, 87/88, 88/89, 89/90, or between anytwo pairs of adjacent residues between 90-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650,650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300,1300-1350, and 1350-1368, relative to SpCas9 of SEQ ID NO: 18, atbetween any two corresponding residues in an amino acid sequence havingat least 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity withSEQ ID NO: 18, or between any two corresponding residues in a variant orequivalent of SpCas9 of any of amino acid sequences SEQ ID NOs. 19-88,or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%,or 99.9% sequence identity with any of SEQ ID NOs: 19-88.

In various embodiments, the split intein sequences can be engineered byfrom the following intein sequences.

NAME SEQUENCE OF LIGAND-DEPENDENT INTEIN 2-4 INTEIN:CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH NC (SEQ ID NO: 472)3-2 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYTNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHN C (SEQ ID NO: 473)30R3-1 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV VHNC (SEQ ID NO: 474)30R3-2 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV VHNC (SEQ ID NO: 475)30R3-3 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV VHNC (SEQ ID NO: 476)37R3-1 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYNPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV VHNC ((SEQ ID NO: 477)37R3-2 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH NC (SEQ ID NO: 478)37R3-3 INTEIN CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH NC (SEQ ID NO: 479)

In various other embodiments, the split intein sequences can be used asfollows:

INTEIN-N INTEIN-C NPU-N NPU-C CLSYETEILTVEYGLLPIGKIVEKRIECIKIATRKYLGKQNVYDIG TVYSVDNNGNIYTQPVAQWHDRGEQ VERDHNFALKNGFIASNEVFEYCLEDGSLIRATKDHKFMTVDG (SEQ ID NO: 452) QMLPIDEIFERELDLMRVDNLPNSGG S(SEQ ID NO: 447)

In various embodiments, the split inteins can be used to separatelydeliver separate portions of a complete PE fusion protein to a cell,which upon expression in a cell, become reconstituted as a complete PEfusion protein through the trans splicing.

In some embodiments, the disclosure provides a method of delivering a PEfusion protein to a cell, comprising:

-   -   (a) constructing a first expression vector encoding an        N-terminal fragment of the PE fusion protein fused to a first        split intein sequence;    -   (b) constructing a second expression vector encoding a        C-terminal fragment of the PE fusion protein fused to a second        split intein sequence;    -   (c) delivering the first and second expression vectors to a        cell,        wherein the N-terminal and C-terminal fragment are reconstituted        as the PE fusion protein in the cell as a result of trans        splicing activity causing self-excision of the first and second        split intein sequences.

The split site in some embodiments can be anywhere in the prime editorfusion, including the napDNAbp domain, the linker, or the reversetranscriptase domain.

In other embodiments, the split site is in the napDNAbp domain.

In still other embodiments, the split site is in the reversetranscriptase or polymerase domain.

In yet other embodiments, the split site is in the linker.

In various embodiments, the present disclosure provides prime editorscomprising a napDNAbp (e.g., a Cas9 domain) and a reverse transcriptasewherein one or both of the napDNAbp and/or the reverse transcriptasecomprise an intein, for example, a ligand-dependent intein. Typicallythe intein is a ligand-dependent intein which exhibits no or minimalprotein splicing activity in the absence of ligand (e.g., smallmolecules such as 4-hydroxytamoxifen, peptides, proteins,polynucleotides, amino acids, and nucleotides). Ligand-dependent inteinsare known, and include those described in U.S. patent application U.S.Ser. No. 14/004,280, published as U.S. 2014/0065711 A1, the entirecontents of which are incorporated herein by reference. In addition, useof split-Cas9 architecture In some embodiments, the intein comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:8-15, 447, 452, 462, and 472-479.

In various embodiments, the napDNAbp domains are smaller-sized napDNAbpdomains as compared to the canonical SpCas9 domain of SEQ ID NO: 18.

The canonical SpCas9 protein is 1368 amino acids in length and has apredicted molecular weight of 158 kilodaltons. The term “small-sizedCas9 variant”, as used herein, refers to any Cas9 variant—naturallyoccurring, engineered, or otherwise—that is less than at least 1300amino acids, or at least less than 1290 amino acids, or than less than1280 amino acids, or less than 1270 amino acid, or less than 1260 aminoacid, or less than 1250 amino acids, or less than 1240 amino acids, orless than 1230 amino acids, or less than 1220 amino acids, or less than1210 amino acids, or less than 1200 amino acids, or less than 1190 aminoacids, or less than 1180 amino acids, or less than 1170 amino acids, orless than 1160 amino acids, or less than 1150 amino acids, or less than1140 amino acids, or less than 1130 amino acids, or less than 1120 aminoacids, or less than 1110 amino acids, or less than 1100 amino acids, orless than 1050 amino acids, or less than 1000 amino acids, or less than950 amino acids, or less than 900 amino acids, or less than 850 aminoacids, or less than 800 amino acids, or less than 750 amino acids, orless than 700 amino acids, or less than 650 amino acids, or less than600 amino acids, or less than 550 amino acids, or less than 500 aminoacids, but at least larger than about 400 amino acids and retaining therequired functions of the Cas9 protein.

In one embodiment, as depicted in Example 20, the specification embracesthe following split-intein PE constructs, which are split betweenresidues 1024 and 1025 of the canonical SpCas9 (SEQ ID NO: 18) (or whichmay be referred to as residues 1023 and 1024, respectively, relative toa Met-minus SEQ ID NO: 18).

First, the amino acid sequence of SEQ ID NO: 18 is shown as follows,indicating the location of the split site between 1024 (“K”) and 1025(“S”) residues:

Description Sequence SEQ ID NO: SpCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN SEQ ID NO: StreptococcusTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR 18, indicated pyogenesKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH with split site M1ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL 1024/1025 in SwissProtRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ bold AccessionTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL The M at No.PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS position 1 is Q99ZW2KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI not necessarily Wild typeLRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL present in thePEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK PE fusionMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH protein inAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN certainSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN embodiments.FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM Thus, theRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI numbering ofECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE the split site isNEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK 1023/1024 inQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG the case thatFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIA the amino acidNLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM sequenceARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV excludes MetENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD at position 1.VDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

In this configuration, the amino acid sequence of N-terminal half (aminoacids 1-1024) is as follows:

(SEQ ID NO: 3877) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIA K .

In this configuration, the amino acid sequence of N-terminal half (aminoacids 1-1023) (where the protein is Met-minus at position 1) is asfollows:

(SEQ ID NO: 3878) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM IA K .

In this configuration, the amino acid sequence of C-terminal half (aminoacids 1024-1368 (or counted as amino acids 1023-1367 in a Met-minusCas9) is as follows:

(SEQ ID NO: 3879) S EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

As shown in Example 20, the PE2 (which is based on SpCas9 of SEQ ID NO:18) construct was split at position 1023/1024 (relative to a Met-minusSEQ ID NO: 18) into two separate constructs, as follows:

SpPE2 split at 1023/1024 N terminal half (SEQ ID NO: 3875)MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY

KRTADGSEFEPKKKRKV 

SpPE2 split at 1023/1024 C terminal half (SEQ ID NO: 3876)

KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG

WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI

The present disclosure also contemplates methods of deliveringsplit-intein prime editors to cells and/or treating cells withsplit-intein prime editors.

In some embodiments, the disclosure provides a method of delivering a PEfusion protein to a cell, comprising:

-   -   (a) constructing a first expression vector encoding an        N-terminal fragment of the PE fusion protein fused to a first        split intein sequence;    -   (b) constructing a second expression vector encoding a        C-terminal fragment of the PE fusion protein fused to a second        split intein sequence;    -   (c) delivering the first and second expression vectors to a        cell,        wherein the N-terminal and C-terminal fragment are reconstituted        as the PE fusion protein in the cell as a result of trans        splicing activity causing self-excision of the first and second        split intein sequences.

In certain embodiments, the N-terminal fragment of the PE fusion proteinfused to a first split intein sequence is SEQ ID NO: 3875, or an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99.9% sequence identity with SEQ ID NO:3875.

In other embodiments, the C-terminal fragment of the PE fusion proteinfused to a first split intein sequence is SEQ ID NO: 3876, or an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99.9% sequence identity with SEQ ID NO:3876.

In other embodiments, the disclosure provides a method of editing atarget DNA sequence within a cell, comprising:

-   -   (a) constructing a first expression vector encoding an        N-terminal fragment of the PE fusion protein fused to a first        split intein sequence;    -   (b) constructing a second expression vector encoding a        C-terminal fragment of the PE fusion protein fused to a second        split intein sequence;    -   (c) delivering the first and second expression vectors to a        cell,        wherein the N-terminal and C-terminal fragment are reconstituted        as the PE fusion protein in the cell as a result of trans        splicing activity causing self-excision of the first and second        split intein sequences.

In certain embodiments, the N-terminal fragment of the PE fusion proteinfused to a first split intein sequence is SEQ ID NO: 3875, or an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99.9% sequence identity with SEQ ID NO:3875.

In other embodiments, the C-terminal fragment of the PE fusion proteinfused to a first split intein sequence is SEQ ID NO: 3876, or an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99.9% sequence identity with SEQ ID NO:3876.

Delivery of PE Ribonucleoprotein Complexes

In this aspect, the prime editors may be delivered by non-viral deliverystrategies involving delivery of a prime editor complexed with a PEgRNA(i.e., a PE ribonucleoprotein complex) by various methods, includingelectroporation and lipid nanoparticles. Methods of non-viral deliveryof nucleic acids include lipofection, nucleofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection is described in e.g., U.S.Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagentsare sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Feigner, WO 91/17424; WO91/16024. Delivery can be to cells (e.g. in vitro or ex vivoadministration) or target tissues (e.g. in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional reference may be made to the following references thatdiscuss approaches for non-viral delivery of ribonucleoproteincomplexes, each of which are incorporated herein by reference.

-   Chen, Sean, et al. “Highly efficient mouse genome editing by CRISPR    ribonucleoprotein electroporation of zygotes.” Journal of Biological    Chemistry (2016): jbc-M116. PubMed-   Zuris, John A., et al. “Cationic lipid-mediated delivery of proteins    enables efficient protein-based genome editing in vitro and in    vivo.” Nature biotechnology 33.1 (2015): 73. PubMed-   Rouet, Romain, et al. “Receptor-Mediated Delivery of CRISPR-Cas9    Endonuclease for Cell-Type-Specific Gene Editing.” Journal of the    American Chemical Society 140.21 (2018): 6596-6603. PubMed.

FIG. 68C provides data showing that various disclosed PEribonucleoprotein complexes (PE2 at high concentration, PE3 at highconcentration and PE3 at low concentration) can be delivered in thismanner.

Delivery of PE by mRNA

Another method that may be employed to deliver prime editors and/orPEgRNAs to cells in which prime editing-based genome editing is desiredis by employing the use of messenger RNA (mRNA) delivery methods andtechnologies. Examples of mRNA delivery methods and compositions thatmay be utilized in the present disclosure including, for example,PCT/US2014/028330, U.S. Pat. No. 8,822,663B2, NZ700688A, ES2740248T3,EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BR112016030852A2,and EP3362461A1, each of which are incorporated herein by reference intheir entireties. Additional disclosure hereby incorporated by referencecan be found in Kowalski et al., “Delivering the Messenger: Advances inTechnologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4):710-728.

In contrast to DNA vector encoding prime editors, the use of RNA asdelivery agent for prime editors has the advantage that the geneticmaterial does not have to enter the nucleus to perform its function. Thedelivered mRNA may be directly translated in the cytoplasm into thedesired protein (e.g., prime editor fusion protein) and nucleic acidproducts (e.g., PEgRNA). However, in order to be more stable (e.g.,resist RNA-degrading enzymes in the cytoplasm), it is in someembodiments necessary to stabilize the mRNA to improve deliveryefficiency. Certain delivery carriers such as cationic lipids orpolymeric delivery carriers can also help protect the transfected mRNAfrom endogenous RNase enzymes that might otherwise degrade thetherapeutic mRNA encoding the desired prime editor fusion proteins. Inaddition, despite the increased stability of modified mRNA, delivery ofmRNA, particularly mRNA encoding full-length protein, to cells in vivoin a manner that allows therapeutic levels of protein production remainsa challenge.

With some exceptions, the intracellular delivery of mRNA is generallymore challenging than that of small oligonucleotides, and it requiresencapsulation into a delivery nanoparticle, in part due to thesignificantly larger size of mRNA molecules (300-5,000 kDa, ˜1-15 kb) ascompared to other types of RNAs (small interfering RNAs [siRNAs], ˜14kDa; antisense oligonucleotides [ASOs], 4-10 kDa).

mRNA must cross the cell membrane in order to reach the cytoplasm. Thecell membrane is a dynamic and formidable barrier to intracellulardelivery. It is made up primarily of a lipid bilayer of zwitterionic andnegatively charged phospholipids, where the polar heads of thephospholipids point toward the aqueous environment and the hydrophobictails form a hydrophobic core.

In some embodiments, the mRNA compositions of the disclosure comprisemRNA (encoding a prime editor and/or PEgRNA), a transport vehicle, andoptionally an agent that facilitates contact with the target cell andsubsequent transfection.

In some embodiments, the mRNA can include one or more modifications thatconfer stability to the mRNA (eg, compared to the wild-type or nativeversion of the mRNA) and is involved in the associated abnormalexpression of the protein. One or more modifications to the wild typethat correct the defect may also be included. For example, the nucleicacids of the invention can include modifications of one or both of a 5′untranslated region or a 3′ untranslated region. Such modifications mayinclude the inclusion of sequences encoding a partial sequence of thecytomegalovirus (CMV) immediate early 1 (IE1) gene, poly A tail, Cap1structure, or human growth hormone (hGH). In some embodiments, the mRNAis modified to reduce mRNA immunogenicity.

In one embodiment, the “prime editor” mRNA in the composition of theinvention can be formulated in a liposome transfer vehicle to facilitatedelivery to target cells. Contemplated transfer vehicles can include oneor more cationic lipids, non-cationic lipids, and/or PEG-modifiedlipids. For example, the transfer vehicle can include at least one ofthe following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003,ICE, HGT5000, or HGT5001. In embodiments, the transfer vehicle comprisescholesterol (chol) and/or PEG modified lipids. In some embodiments, thetransfer vehicle comprises DMG-PEG2K. In certain embodiments, thetransfer vehicle has the following lipid formulation: C12-200, DOPE,chol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE,chol, DMG-PEG2K, HGT5001, DOPE, chol, one of DMG-PEG2K.

The present disclosure also provides compositions and methods useful forfacilitating transfection of target cells with one or more PE-encodingmRNA molecules. For example, the compositions and methods of the presentinvention contemplate the use of targeting ligands that can increase theaffinity of the composition for one or more target cells. In oneembodiment, the targeting ligand is apolipoprotein B or apolipoproteinE, and the corresponding target cells express low density lipoproteinreceptors and thus promote recognition of the targeting ligand. A vastnumber of target cells can be preferentially targeted using the methodsand compositions of the present disclosure. For example, contemplatedtarget cells include hepatocytes, epithelial cells, hematopoietic cells,epithelial cells, endothelial cells, lung cells, bone cells, stem cells,mesenchymal cells, nerve cells, heart cells, adipocytes, vascular smoothmuscle Includes cells, cardiomyocytes, skeletal muscle cells, betacells, pituitary cells, synovial lining cells, ovarian cells, testiscells, fibroblasts, B cells, T cells, reticulocytes, leukocytes,granulocytes, and tumor cells However, it is not limited to these.

In some embodiments, the PE-encoding mRNA may optionally have chemicalor biological modifications which, for example, improve the stabilityand/or half-life of such mRNA or which improve or otherwise facilitateprotein production. Upon transfection, a natural mRNA in thecompositions of the invention may decay with a half-life of between 30minutes and several days. The mRNAs in the compositions of thedisclosure may retain at least some ability to be translated, therebyproducing a functional protein or enzyme. Accordingly, the inventionprovides compositions comprising and methods of administering astabilized mRNA. In some embodiments, the activity of the mRNA isprolonged over an extended period of time. For example, the activity ofthe mRNA may be prolonged such that the compositions of the presentdisclosure are administered to a subject on a semi-weekly or bi-weeklybasis, or more preferably on a monthly, bi-monthly, quarterly or anannual basis. The extended or prolonged activity of the mRNA of thepresent invention is directly related to the quantity of protein orenzyme produced from such mRNA. Similarly, the activity of thecompositions of the present disclosure may be further extended orprolonged by modifications made to improve or enhance translation of themRNA. Furthermore, the quantity of functional protein or enzyme producedby the target cell is a function of the quantity of mRNA delivered tothe target cells and the stability of such mRNA. To the extent that thestability of the mRNA of the present invention may be improved orenhanced, the half-life, the activity of the produced protein or enzymeand the dosing frequency of the composition may be further extended.

Accordingly, in some embodiments, the mRNA in the compositions of thedisclosure comprise at least one modification which confers increased orenhanced stability to the nucleic acid, including, for example, improvedresistance to nuclease digestion in vivo. As used herein, the terms“modification” and “modified” as such terms relate to the nucleic acidsprovided herein, include at least one alteration which preferablyenhances stability and renders the mRNA more stable (e.g., resistant tonuclease digestion) than the wild-type or naturally occurring version ofthe mRNA. As used herein, the terms “stable” and “stability” as suchterms relate to the nucleic acids of the present invention, andparticularly with respect to the mRNA, refer to increased or enhancedresistance to degradation by, for example nucleases (i.e., endonucleasesor exonucleases) which are normally capable of degrading such mRNA.Increased stability can include, for example, less sensitivity tohydrolysis or other destruction by endogenous enzymes (e.g.,endonucleases or exonucleases) or conditions within the target cell ortissue, thereby increasing or enhancing the residence of such mRNA inthe target cell, tissue, subject and/or cytoplasm. The stabilized mRNAmolecules provided herein demonstrate longer half-lives relative totheir naturally occurring, unmodified counterparts (e.g. the wild-typeversion of the mRNA). Also contemplated by the terms “modification” and“modified” as such terms related to the mRNA of the present inventionare alterations which improve or enhance translation of mRNA nucleicacids, including for example, the inclusion of sequences which functionin the initiation of protein translation (e.g., the Kozak consensussequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

In some embodiments, the mRNAs used in the compositions of thedisclosure have undergone a chemical or biological modification torender them more stable. Exemplary modifications to an mRNA include thedepletion of a base (e.g., by deletion or by the substitution of onenucleotide for another) or modification of a base, for example, thechemical modification of a base. The phrase “chemical modifications” asused herein, includes modifications which introduce chemistries whichdiffer from those seen in naturally occurring mRNA, for example,covalent modifications such as the introduction of modified nucleotides,(e.g., nucleotide analogs, or the inclusion of pendant groups which arenot naturally found in such mRNA molecules).

Other suitable polynucleotide modifications that may be incorporatedinto the PE-encoding mRNA used in the compositions of the disclosureinclude, but are not limited to, 4′-thio-modified bases:4′-thio-adenosine, 4′-thio-guanosine, 4′-thio-cytidine, 4′-thio-uridine,4′-thio-5-methyl-cytidine, 4′-thio-pseudouridine, and4′-thio-2-thiouridine, pyridin-4-one ribonucleoside, 5-aza-uridine,2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, andcombinations thereof. The term modification also includes, for example,the incorporation of non-nucleotide linkages or modified nucleotidesinto the mRNA sequences of the present invention (e.g., modifications toone or both of the 3′ and 5′ ends of an mRNA molecule encoding afunctional protein or enzyme). Such modifications include the additionof bases to an mRNA sequence (e.g., the inclusion of a poly A tail or alonger poly A tail), the alteration of the 3′ UTR or the 5′ UTR,complexing the mRNA with an agent (e.g., a protein or a complementarynucleic acid molecule), and inclusion of elements which change thestructure of an mRNA molecule (e.g., which form secondary structures).

In some embodiments, PE-encoding mRNAs include a 5′ cap structure. A 5′cap is typically added as follows: first, an RNA terminal phosphataseremoves one of the terminal phosphate groups from the 5′ nucleotide,leaving two terminal phosphates; guanosine triphosphate (GTP) is thenadded to the terminal phosphates via a guanylyl transferase, producing a5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is thenmethylated by a methyltransferase. Examples of cap structures include,but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A andG(5′)ppp(5′)G. Naturally occurring cap structures comprise a 7-methylguanosine that is linked via a triphosphate bridge to the 5′-end of thefirst transcribed nucleotide, resulting in a dinucleotide cap ofm7G(5′)ppp(5′)N, where N is any nucleoside. In vivo, the cap is addedenzymatically. The cap is added in the nucleus and is catalyzed by theenzyme guanylyl transferase. The addition of the cap to the 5′ terminalend of RNA occurs immediately after initiation of transcription. Theterminal nucleoside is typically a guanosine, and is in the reverseorientation to all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.

Additional cap analogs include, but are not limited to, a chemicalstructures selected from the group consisting of m7GpppG, m7GpppA,m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog(e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG),dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reversecap analogs (e.g., ARCA; m7,2′OmeGpppG, m72′dGpppG, m7,3′OmeGpppG,m7,3′dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity,J. et al., “Novel ‘anti-reverse’ cap analogs with superior translationalproperties”, RNA, 9: 1108-1122 (2003)).

Typically, the presence of a “tail” serves to protect the mRNA fromexonuclease degradation. A poly A or poly U tail is thought to stabilizenatural messengers and synthetic sense RNA. Therefore, in certainembodiments a long poly A or poly U tail can be added to an mRNAmolecule thus rendering the RNA more stable. Poly A or poly U tails canbe added using a variety of art-recognized techniques. For example, longpoly A tails can be added to synthetic or in vitro transcribed RNA usingpoly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14:1252-1256). A transcription vector can also encode long poly A tails. Inaddition, poly A tails can be added by transcription directly from PCRproducts. Poly A may also be ligated to the 3′ end of a sense RNA withRNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed.,ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor LaboratoryPress: 1991 edition)).

Typically, the length of a poly A or poly U tail can be at least about10, 50, 100, 200, 300, 400 at least 500 nucleotides. In someembodiments, a poly-A tail on the 3′ terminus of mRNA typically includesabout 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosinenucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3′poly(C) tail structure. A suitable poly-C tail on the 3′ terminus ofmRNA typically include about 10 to 200 cytosine nucleotides (e.g., about10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides,about 20 to 70 cytosine nucleotides, about 20 to 60 cytosinenucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tailmay be added to the poly-A or poly U tail or may substitute the poly-Aor poly U tail.

PE-encoding mRNAs according to the present disclosure may be synthesizedaccording to any of a variety of known methods. For example, mRNAsaccording to the present invention may be synthesized via in vitrotranscription (IVT). Briefly, IVT is typically performed with a linearor circular DNA template containing a promoter, a pool of ribonucleotidetriphosphates, a buffer system that may include DTT and magnesium ions,and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase),DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditionswill vary according to the specific application.

In embodiments involving mRNA delivery, the ratio of the mRNA encodingthe PE fusion protein to the PEgRNA may be important for efficientediting. In certain embodiments, the weight ratio of mRNA (encoding thePE fusion protein) to PEgRNA is 1:1. In certain other embodiments, theweight ratio of mRNA (encoding the PE fusion protein) to PEgRNA is 2:1.In still other embodiments, the weight ratio of mRNA (encoding the PEfusion protein) to PEgRNA is 1:2. In still further embodiments, theweight ratio of mRNA (encoding the PE fusion protein) to PEgRNA isselected from the group consisting of about 1:1000, 1:900; 1:800; 1:700;1:600; 1:500; 1:400; 1:300; 1:200; 1:100; 1:90; 1:80; 1:70; 1:60; 1:50;1:40; 1:30; 1:20; 1:10; and 1:1. In other embodiments, the weight ratioof mRNA (encoding the PE fusion protein) to PEgRNA is selected from thegroup consisting of about 1:1000, 1:900; 800:1; 700:1; 600:1; 500:1;400:1; 300:1; 200:1; 100:1; 90:1; 80:1; 70:1; 60:1; 50:1; 40:1; 30:1;20:1; 10:1; and 1:1.

J. Use of Prime Editing for Identifying Off-Target Editing in anUnbiased Manner

Like other genome editors, there exists some risk that PE may introduceits programmed genetic alterations at unintended sites around thegenome, i.e., “off-target” sites. However, there are currently nodescribed methods to detect off-target editing with prime editors. Suchmethods would allow the identification of potential sites of off-targetediting using prime editors.

The key concept of this aspect is the idea of using prime editing toinsert the same adapter sequence and/or primer binding site at on-targetand off-target sites, templated from the same PEgRNA, to enable therapid identification of genomic off-target modification sites ofnapDNAbp nucleases or prime editors. This method is distinguished fromother techniques that identify nuclease off-target sites because theadapter and/or primer binding sequence is inserted in the same event asDNA binding and nicking by the napDNAbp, simplifying the downstreamprocessing.

FIG. 33 illustrates the basic principle of off-target identification.The figure is a schematic showing PEgRNA design for primer bindingsequence insertions and primer binding insertion into genomic DNA usingprime editing for determining off-target editing. In this embodiment,prime editing is conducted inside a living cell, a tissue, or an animalmodel. As a first step, an appropriate PEgRNA is designed. The topschematic shows a exemplary PEgRNA that may be used in this aspect. Thespacer in the PEgRNA (labeled as “protospacer”) is complementary to oneof the strands of the genomic target. The PE:PEgRNA complex (i.e., thePE complex) installs a single stranded 3′ end flap at the nick sitewhich contains the encoded primer binding sequence and the region ofhomology (coded by the homology arm of the PEgRNA) that is complementaryto the region just downstream of the cut site (in bold). Through flapinvasion and DNA repair/replication processes, the synthesized strandbecomes incorporated into the DNA, thereby installing the primer bindingsite. This process can occur at the desired genomic target, but also atother genomic sites that might interact with the PEgRNA in an off-targetmanner (i.e., the PEgRNA guides the PE complex to other off-target sitesdue to the complementarity of the spacer region to other genomic sitesthat are not the intended genomic site). Thus, the primer bindingsequence may be installed not only at the desired genomic target, but atoff-target genomic sites elsewhere in the genome. In order to detect theinsertion of these primer binding sites at both the intended genomictarget sites and the off-target genomic sites, the genomic DNA (post-PE)can be isolated, fragmented, and ligated to adapter nucleotides. Next,PCR may be carried out with PCR oligonucleotides that anneal to theadapters and to the inserted primer binding sequence to amplifyon-target and off-target genomic DNA regions into which the primerbinding site was inserted by PE. High throughput sequencing then may beconducted, as well as sequence alignments, to identify the insertionpoints of PE-inserted primer binding sequences at either the on-targetsite or at off-target sites.

Thus, FIG. 33 illustrates one aspect regarding the identification ofoff-target editing sites when editing inside a living cell, in tissueculture or animal models. To conduct this method, a PEgRNA is generatedthat has an identical spacer to the final desired prime editor (and, iflooking at prime editing off-targets, an identical primer-binding sitesequence to the final desired editor), but includes the necessarysequences to install an adapter or primer binding site after reversetranscription by prime editing. In vivo editing is conducted using aprime editor or RT-fused nuclease, and isolate genomic DNA. The genomicDNA is fragmented by enzymatic or mechanical means and append adifferent adapter to sites of DNA fragmentation. PCR is used to amplifyfrom one adapter to the adapter installed via PEgRNA. The resultingproduct is deep-sequenced to identify all modified sites.

In another aspect, evaluation of off-target editing by PE may beconducted in vitro. In this aspect, PE may be used during in vitromodification of genomic DNA identification of off-target editing sitesusing in vitro modification of genomic DNA. To conduct this method,ribonucleoprotein (RNP) of purified prime editor fusion protein and aPEgRNA (i.e., the PE complex) is assembled that is configured to installan adapter or primer binding sequence at a target site, but is otherwisethe same as the PEgRNA of interest. This RNP (i.e., PE complex) isincubated with extracted genomic DNA before or after fragmentation ofthe DNA. After fragmentation, different adapters sequences are ligatedto the ends of the fragment DNA. PCR is used to amplify those genomicsites that span the inserted adapter sequence (i.e., inserted by EP) andthe adapters ligated to fragment ends. High throughput sequencingbetween the adapters sequences can identify genomic sites ofmodification that are on-target and off-target. This in vitro editingmethod should enhance the sensitivity of detection because cellular DNArepair will not eliminate the reverse-transcribed DNA adapter added bythe prime editor.

These methods could be used to identify off-target editing for any primeeditor, or any genome editor that uses a guide RNA to recognize a targetcut site (most Cas nucleases).

These methods could be applied to all genetic diseases for which genomeeditors are considered for use in treatment.

Exemplary adapter and/or primer binding sequences that may be installedby PE include, but are not limited to:

ADAPTER 1 5′-CGGTGGACCGATGATCT-3′(SEQ ID NO: 177)  ADAPTER 25′-GCCACCTGGCTACTAGA-3′(SEQ ID NO: 178)  ADAPTER 35′-AGATCATCGGTCCACCG-3′(SEQ ID NO: 179) ADAPTER 45′-TCTAGTAGCCAGGTGGC-3′(SEQ ID NO: 180)

These adapter and/or primer binding sequences may also be used in theligation step after genomic DNA fragmentation as outlined above.

Exemplary PEgRNA designs that illustrate the use of the herein describedmethod to evaluate off-target editing, and their edit target locus, areas follows:

HEK3 TEST LOCUS OFF- GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAGAAATATARGET DISCOVERY: GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCTGCCATCA CGGTG GACCGATGATCTCGTGCTCAGTCTG (SEQ ID NO: 181) HEK4 TEST LOCUS OFF-GGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAAT TARGET DISCOVERY:AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACACAGCACCAGAG TCTCCGCTTTAACCCCCAGCCACCTGGCTACTAGA CC TCCAGCC (SEQ ID NO: 182) SICKLE CELLGCATGGTGCACCTGACTCCTGGTTTTAGAGCTAGAAA CORRECTION OFF-TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG TARGET DISCOVERY:AAAAAGTGGCACCGAGTCGGTGCAGACTTCTCTTCA G GCCACCTGGCTACTAGAGAGTCAGGTGCAC (SEQ ID NO: 183) KEY PBS Spacer sgRNA scaffoldDNA synthesis  template Adapter PBS

K. Use of Prime Editing for Insertion of Inducible Dimerization Domains

The prime editors described herein may also be used to installdimerization domains into one or more protein targets. The dimerizationdomains may facilitate inducible regulation of the activity associatedwith the dimerization of the one or more protein targets via a linkingmoiety (e.g., a small molecule, peptide, or protein) that binds in abi-specific manner. In various aspects, the dimerization domains, wheninstalled on different proteins (e.g., the same type or differentproteins), each bind to the same bi-specific moiety (e.g., a bi-specificsmall molecule, peptide, or polypeptide having a least two regions thatseparately bind to the dimerization domains), thereby causing thedimerization of the proteins through their common interaction to thebi-specific ligand. In this manner, the bi-specific ligand functions asan “inducer” of dimerization of two proteins. In some cases, thebi-specific ligand or compound will have two targeting moieties that arethe same. In other embodiments, the bi-specific ligand or compound willhave targeting moieties that are each different from the other. Thebi-specific ligand or compound having the same two targeting moietieswill be able to target the same dimerization domain installed ondifferent protein targets. The bi-specific ligand or compound havingdifferent targeting moieties will be able to target differentdimerization domains installed on different protein targets.

As used herein, the term “dimerization domain” refers to aligand-binding domain that binds to a binding moiety of a bi-specificligand. A “first” dimerization domain binds to a first binding moiety ofa bi-specific ligand and a “second” dimerization domain binds to asecond binding moiety of the same bi-specific ligand. When the firstdimerization domain is fused to a first protein (e.g., via PE, asdiscussed herein) and the second dimerization domain (e.g., via PE, asdiscussed herein) is fused to a second protein, the first and secondprotein dimerize in the presence of a bi-specific ligand, wherein thebi-specific ligand has at least one moeity that binds to the firstdimerization domain and at least another moiety that binds to the seconddimerization domain.

The term “bi-specific ligand” or “bi-specific moiety,” as used herein,refers to a ligand that binds to two different ligand-binding domains.In various embodiments, the bi-specific moiety itself is a dimer of twoof same or two different chemical moieties, wherein each moietyspecifically and tightly binds to a dimerization domain. In certainembodiments, the ligand is a small molecule compound, or a peptide, or apolypeptide. In other embodiments, ligand-binding domain is a“dimerization domain,” which can be install as a peptide tag onto aprotein. In various embodiments, two proteins each comprising the sameor different dimerization domains can be induced to dimerize through thebinding of each dimerization domain to the bi-specific ligand. Thesemolecules may also be referred to as “chemical inducers of dimerization”or CIDs. In addition, the bi-specific ligands may be prepared bycoupling (e.g., through standardize chemical linkages) two of the samemoieties together, or two different moieties together, wherein eachmoiety tightly and specifically binds to a dimerization domain.

In various aspects, the dimerization domains installed by PE can be thesame or different.

For example, the dimerization domains can be FKBP12, which has thefollowing amino acid sequence:

FKBP12 MGVQVETISPGDGRTFPKRGQTCVVHYTG MLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATG HPGIIPPHATLVFDVELLKLE (SEQ ID NO: 488)

In another example, the dimerization domain can be a mutant of FKBP12referred to as FKBP12-F36V, a mutant of FKBP12 with an engineered holethat binds a synthetic bumped FK506 mimic (2, FIG. 3 )¹⁰⁷:

FKBP12-F36V MGVQVETISPGDGRTFPKRGQTCVVHYTG MLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGAT GHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 489)

In another example, the dimerization domain can be cyclophilin, asfollows:

CYCLOPHILIN MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGF MCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKT EWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE (SEQ ID NO: 490)

In various embodiments, the amino acid sequences of these dimerizationdomains may be altered in order to optimize binding or to improvebinding orthogonality to native targets. The nucleic acid sequences ofthe genes encoding small-molecule binding proteins may be altered inorder to optimize the efficiency of the PE process, such as by reducingPEgRNA secondary structure.

Other examples of suitable dimerization domains and a cognate smallmolecule compound which binds thereto are provided as follows. Note thatthe cognate small molecule compound could be coupled (e.g., via achemical linker) to a second small molecule compound (either the samecompound or a different compound) in order to form a bi-specific ligandthat may bind two dimerization domains. In some cases, such as FK506 andcyclosporin A, dimerization of each (e.g., FK506-FK506 or cyclosporinA-cyclosporin A) reduces or eliminates immunosuppressive activity of themonomeric compounds.

SMALL MOLECULE—BINDS TO THE DIMERIZATION DOMAIN—A DIMER OF THESE MOLECULES WOULD CONSTITUTE ABI-SPECIFIC LIGAND THAT WOULD BIND TWO DIMERIZATION DOMAINSDIMERIZATION DOMAIN(S) FK506  

  FK-506 FKBP12 Kd 0.4 nM PNAS 1990, 87, 9231.TARGETS: FKBP12 + CALCINEURIN AMINO ACID SEQUENCE OF FKBP12:MGVQVETISPGDGRTFPKRGQTCV VHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQ RAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 491) CALCINEURIN: CYCLOSPORIN A  

  TARGETS: CYCLOPHILIN + CALCINEURIN AMINO ACID SEQUENCE OFHUMAN CYCLOPHILIN A: MVNPTVFFDIAVDGEPLGRVSFELF ADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTRHNGTG GKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDG KHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE (SEQ ID NO: 490) AMINO ACID SEQUENCE OFHUMAN CYCLOPHILIN B: MLRLSERNMKVLLAAALIAGSVFF LLLPGPSAADEKKKGPKVTVKVYFDLRIGDEDVGRVIFGLFGKTVPKTV DNFVALATGEKGFGYKNSKFHRVIKDFMIQGGDFTRGDGTGGKSIYGER FPDENFKLKHYGPGWVSMANAGKDTNGSQFFITTVKTAWLDGKHVVF GKVLEGMEVVRKVESTKTDSRDKPLKDVIIADCGKIEVEKPFAIAKE (SEQ ID NO: 493) AMINO ACID SEQUENCE OFMURINE CYCLOPHILIN C: MSPGPRLLLPAVLCLGLGALVSSSG SSGVRKRGPSVTDKVFFDVRIGDKDVGRIVIGLFGNVVPKTVENFVALA TGEKGYGYKGSIFHRVIKDFMIQGGDFTARDGTGGMSIYGETFPDENFKL KHYGIGWVSMANAGPDTNGSQFFITLTKPTWLDGKHVVFGKVLDGMT VVHSIELQATDGHDRPLTDCTIVNSGKIDVKTPFVVEVPDW (SEQ ID NO: 494) AP1867  

  AP1867 FK-506 mimic FKBP12 F36V Kd 94 pM FKBP12 Kd 67 nMPNAS 1998, 95, 10437. TARGET(S): FKBP12 AMINO ACID SEQUENCE OFFKBP12-F36V MGVQVETISPGDGRTFPKRGQTCV VHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVG QRAKLT ISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 495) METHOTREXATE  

  methotrexate Human DHFR Kd < 10 nM J. Biol. Chem. 1988, 263, 10304.E. coli DHFR Kd 9.5 nM PNAS 2002, 99, 13481.TARGET(S): DIHYDROFOLATE REDUCTASE AMINO ACID SEQUENCE OFHUMAN DIHYDROFOLATE REDUCTASE MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMTTTSSVEGKQ NLVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDAL KLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESD TFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND (SEQ ID NO: 496) TRIMETHOPRIM  

  trimethoprim E. coli DHFR K_(I )1.3 nM Biochemistry 1982, 21, 5068.TARGET(S): DIHYDROFOLATE REDUCTASE AMINO ACID SEQUENCE FOR E.COLI DIHYDROFOLATE REDUCTASE MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWE SIGRPLPGRKNIILSSQPGTDDRVTWVKS VDEAIAACGDVPEIMVIGGGRV YEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNS HSYCFEILERR (SEQ ID NO: 497) DEXAMETHOSONE  

  dexamethasone Human GR Kd 4.6 nM Mol. Endocrin. 1999, 13, 1855.AMINO ACID SEQUENCE FOR HUMAN GLUCOCORTICOID RECEPTORMDSKESLTPGREENPSSVLAQERGD VMDFYKTLRGGATVKVSASSPSLAVASQSDSKQRRLLVDFPKGSVSNA QQPDLSKAVSLSMGLYMGETETKVMGNDLGFPQQGQISLSSGETDLKLL EESIANLNRSTSVPENPKSSASTAVS AAPTEKEFPKTHSDVSSEQQHLKGQTGTNGGN VKLYTTDQSTFDILQDLEFSSGSPGKETNESPWRSDLLIDENCLLSPLAG EDDSFLLEGNSNEDCKPLILPDTKPKIKDNGDLVLSSPSNVTLPQVKTEK EDFIELCTPGVIKQEKLGTVYCQASFPGANIIGNKMSAISVHGVSTSGGQ MYHYDMNTASLSQQQDQKPIFNVIPPIPVGSENWNRCQGSGDDNLTSLG TLNFPGRTVFSNGYSSPSMRPDVSSPPSSSSTATTGPPPKLCLVCSDEASG CHYGVLTCGSCKVFFKRAVEGQHNYLCAGRNDCIIDKIRRKNCPACRYR KCLQAGMNLEARKTKKKIKGIQQATTGVSQETSENPGNKTIVPATLPQLT PTLVSLLEVIEPEVLYAGYDSSVPDSTWRIMTTLNMLGGRQVIAAVKWA KAIPGFRNLHLDDQMTLLQYSWMFLMAFALGWRSYRQSSANLLCFAPD LIINEQRMTLPCMYDQCKHMLYVSSELHRLQVSYEEYLCMKTLLLLSSV PKDGLKSQELFDEIRMTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDS MHEVVENLLNYCFQTFLDKTMSIEFPEMLAEIITNQIPKYSNGNIKKLLF HQK (SEQ ID NO: 498)

  RAPAMYCIN AMINO ACID SEQUENCE OF FKBP12: MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFK FMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHAT LVFDVELLKLE (SEQ ID NO: 491)

Other examples of naturally occurring bifunctional molecules and theirdual target receptors are as follows. Prime editing may be used toinstall the dual target receptors into different proteins. Once thedifferent proteins are modified by PE to contain a bifunctional moleculereceptor, the bifunctional molecules may be introduced, thereby causingthe dimerization of the proteins modified to comprise the differentdimerization domains. Examples of pairings of (1) a biofunctionalmolecule and (2) their dual target receptors are as follows:

TARGET  RECEPTORS  OF THE BIOFUNCTIONAL NATURALLY OCCURRING BIOFUNCTIONAL MOLECULES MOLECULE

  auxin Target  receptor 1:  auxin  receptor Target  receptor 2: TIR1 E3  ligase

  methyl jasmonate Target  receptor 1:  JAZ receptor Target receptor 2:  Col1 E3  ligase brefeldin A  

target  receptor 1:  GBF1 target  receptor 2:  GTPase  Arf1pabscisic acid  

target  receptor 1:  PYR receptor target  receptor 2:  phospho- protein phos- phatase 2C Forskolin  

target  receptor 1:  adenylyl  cyclase  monomers target  receptor 2: adenylyl  cyclase  monomers fusicoccin A  

target  receptor 1:  14-3-3  proteins target  receptor 2:  H⁺-ATPaseRapamycin  

target  receptor 1:  FKBP12 target  receptor 2:  mTOR sanglifehrin A  

  Sanglifehrin A (SFA) target  receptor 1:  cyclophilin target receptor 2:  IMP  dehydro- genase 2 cyclosporin A  

target  receptor 1:  cyclophilin target  receptor 2:  calcineurin

Examples of other bifunctional molecules that can be used with thisaspect of prime editing are as follows:

Synstab A, paclitaxel, and discodermolide are microtubule stabilizers.Thus, these compounds could be used to dimerize proteins modified by PEto comprise microtubule proteins. GNE-0011, ARV-825, and dBET comprise aBRD4 binding motif and a CRBN binding motif. Thus, these compounds couldbe used to dimerize proteins modified by PE to comprise these targetingdomains.

The PEgRNAs for installing dimerization domains may comprising thefollowing structures (in reference to FIG. 3D): 5′-[spacer]-[gRNAcore]-[extension arm]-3′, wherein the extension arm comprises5′-[homology arm]-[edit template]-[primer binding site]-3′; or5′-[extension arm]-[spacer]-[gRNA core]-3′, wherein the extension armcomprises 5′-[homology arm]-[edit template]-[primer binding site]-3′,and wherein with either configuration the “edit template” comprises anucleotide sequence of a dimerization domain.

In one example, the PEgRNA for insertion of the FKBP12 dimerizationdomain at the C-terminal end of human insulin receptor (spacerunderlined, gRNA core plain, flap homology bold, FKBP12 insertion initalics, annealing region bold italics):

PEGRNA FOR CACGGUAGGCACUGUUAGGAGUUUUAG INSTALLINGAGCUAGAAAUAGCAAGUUAAAAUAAG FKBP12 IN GCUAGUCCGUUAUCAACUUGAAAAAGU HUMANGGCACCGAGUCGGUGCUUGCCUCGGU INSULIN CCAAUCCUUCC GGAGUGCAGGUGGAAA RECEPTORCCAUCUCCCCAGGAGACGGGCGCACCUU CCCCAAGCGCGGCCAGACCUGCGUGGUGCACUACACCGGGAUGCUUGAAGAUGGAA AGAAAUUUGAUUCCUCCCGGGACAGAAACAAGCCCUUUAAGUUUAUGCUAGGCAAG CAGGAGGUGAUCCGAGGCUGGGAAGAAGGGGUUGCCCAGAUGAGUGUGGGUCAGA GAGCCAAACUGACUAUAUCUCCAGAUUAUGCCUAUGGUGCCACUGGGCACCCAGGC AUCAUCCCACCACAUGCCACUCUCGUCUUCGAUGUGGAGCUUCUAAAACUGGAA

(SEQ ID NO: 499)

In another example, the PEgRNA for insertion of the FKBP12 dimerizationdomain at the HEK3 locus (for optimization):

PEGRNA FOR GGCCCAGACTGAGCACGTGAGTTTTAGA INSTALLINGGCTAGAAATAGCAAGTTAAAATAAGGC FKBP12 IN HEK3 TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGAGGAAGCAGGG CTTCCTTTCCTCTGCCATCATTCCAGTTTTAGAAGCTCCACATCGAAGACGAGAGT GGCATGTGGTGGGATGATGCCTGGGTGCCCAGTGGCACCATAGGCATAATCTGGAG ATATAGTCAGTTTGGCTCTCTGACCCACACTCATCTGGGCAACCCCTTCTTCCCAG CCTCGGATCACCTCCTGCTTGCCTAGCATAAACTTAAAGGGCTTGTTTCTGTCCCG GGAGGAATCAAATTTCTTTCCATCTTCAAGCATCCCGGTGTAGTGCACCACGCAGG TCTGGCCGCGCTTGGGGAAGGTGCGCCCGTCTCCTGGGGAGATGGTTTCCACCTGC ACTCCCGTGCTCAGTCTG (SEQ ID NO: 500)

The target proteins for installing dimerization domains are notparticularly limited; however, it is advantageous their dimerization(once modified by PE) in the presence of a bi-specific ligand producessome advantageous biological effect, e.g., a signaling pathway,decreased immunoresponsiveness, etc. In various aspects, the targetproteins that are to be dimerized through the PE-dependent installationof dimerization domains can be the same protein or different proteins.Preferably, the proteins, when dimerized, trigger one or more downstreambiological cascades, e.g., a signal transduction cascade,phosphorylation, etc. Exemplary target proteins into which PE may beused to install dimerization domains, includes, but are not limited to:

MEMBRANDE- KINASE BOUND DOMAIN CID SIGNALING RECEPTOR FUSED TO EMPLOYEDCASCADE REFERENCE T-CELL FKBP12 FK1012 T-CELL SCIENCE 262, 1019-24RECEPTOR (FK506 RECEPTOR (1993) DIMER) SIGNALING CHEM. BIOL. 1, 163-172(1994). FAS RECEPTOR MURINE CYCLOSPORIN FAS CHEM. BIOL. 3, 731-738CYCLOPHILIN A PATHWAY (1996). C DIMER FOR APOPTOSIS INSULIN FKBP12FK1012 INSULIN CURR. BIOL. 8, 11-18 RECEPTOR (FK506 SIGNALING (1998).DIMER) PLATELET- FKBP12 FK1012 PDGF CURR. BIOL. 8, 11-18 DERIVED (FK506MESODERM (1998). GROWTH DIMER) FORMATION FACTOR (PDGF) SIGNALING BETAERYTHROPOIE FKBP12 FK1012 EPOR- PROC. NATL. ACAD. SCI. TIN RECEPTOR(FK506 MEDIATED 94, 3076-3081 (2002). (EPOR) DIMER) PROLIFERATIVESIGNALING

In one aspect, prime editors described herein may be used to installsequences encoding dimerization domains into one or more genes encodingtarget proteins of interest in a living cell or patient. This may bereferred to as the “prime editing-CID system,” wherein the CID is thebi-specific ligand that induced dimerization of target proteins, eachfused to a dimerization domain installed by PE. This edit alone shouldhave no physiological effect. Upon administration of a bi-specificligand, which typically is a dimeric small molecule that cansimultaneously bind to two dimerization domains each of which is fusedto a copy of the target protein, the bi-specific ligand causesdimerization of the targeted protein. This target protein dimerizationevent then induces a biological signaling event, such as erythropoiesisor insulin signaling. A new method to place dimerization-inducedbiological processes, such as receptor signaling, under control of aconvenient small-molecule drug (i.e., the bi-specific ligand) by thegenomic integration of genes encoding small-molecule binding proteins(i.e., the dimerization domains) with prime editing is described herein.

Protein dimerization is a ubiquitous biological process. Notably,homodimerization of many membrane-bound receptors is known to initiatesignaling cascades, often with profound biological consequences. Anumber of small-molecule natural products approved for use as drugs actas chemical inducers of protein dimerization as part of their mechanismof action.⁹² For example, FK506 binds tightly to FKBP12, and theresulting small molecule-protein complex then binds the phosphatasecalcineurin, thereby inhibiting a step in T cell receptor signaling.⁹³Likewise, cyclosporin A induces dimerization of cyclophilin andcalcineurin, and rapamycin induces dimerization of FKBP andmTOR.^(93,94)

In one embodiment, leveraging the selective, high-affinity binding ofthe FK506:FKBP12 and cyclosporin A:cyclophilin small molecule:proteinbinding interaction, synthetic chemical inducers of dimerization havealso been developed. In an example, a small molecule comprised of twounits of FK506, termed FK1012, was shown to effect signal transductionwhen the cytoplasmic domains of signaling receptors were tagged withFKBP12.⁹⁵ Chemical inducers of dimerization (CIDs) have since been usedto control a number of signaling pathways.⁹⁶⁻¹⁰³

While useful tools for studying biological processes, one challengefacing synthetic CIDs for therapeutic applications is that introductionof the FKBP12- or cyclophilin-target protein chimeras into patients ischallenging.

The present disclosure brings together two concepts to create apreviously inaccessible therapeutic process. The first concept is primeediting, described herein, which allows for precise genome editing,including targeted insertions, in living cells. The second concept ischemical-induced dimerization, a powerful tool that has enabledsmall-molecule control over signaling and oligomerization processes incell culture.

Specific cases in which chemical control over protein dimerization mayhave had a beneficial therapeutic effect have been identified.

The insulin receptor is a heterotetrameric transmembrane protein thatresponds to insulin binding to the extracellular domain byphosphorylation of the cytoplasmic kinase domain.¹⁰⁴ An engineeredchimeric protein composed of a membrane-localization component, theC-terminal kinase domain of the insulin receptor, and three copies ofFKBP12 responds to FK1012 and initiates the insulin response in cellculture.⁹⁹ Similarly, it is expected that the fusion of FKBP12 to theC-terminal end of the kinase domain of the native insulin receptor inpatient cells should allow for FK1012-dependent phosphorylation andinitiation of the insulin signaling cascade. This system could replaceor complement insulin use in patients who cannot make insulin (e.g.,type-1 diabetics), or who respond weakly to insulin (e.g., type-2diabetics).

Additionally, erythropoietin stimulates erythrocyte proliferation bybinding to the erythropoietin receptor (EpoR), either inducingdimerization or a conformational change in a preformed receptor dimerwhich results in activation of the Jak/STAT signaling cascade.¹⁰⁵ It hasbeen demonstrated that FK1012-induced oligomerization of themembrane-anchored cytoplasmic domain of EpoR tagged with FKBP12 issufficient to initiate the signaling Jak/STAT signaling cascade andpromote cell proliferation.¹⁰⁶ It is anticipated that fusing FKBP12 tonative EpoR by prime editing in patient cells will allow forFK1012-induced control over erythrocyte proliferation (erythropoiesis).This system could be used to trigger red blood cell growth in anemicpatients. FK1012-inducible EpoR could also be employed as an in vivoselectable marker for blood cells that have undergone ex vivoengineering.

In principle, any receptor tyrosine kinase could be viable target for aprime editing-CID therapeutic. The table below includes a list of allreceptor tyrosine kinases in the human genome.¹¹⁰

PROT Family Receptor Synonyms NT Accession Accession Chromosome ALK ALKKi1 NM_004304 NP_004295 2p23 family LTK TYK1 NM_002344 NP_00233515q15.1-q21.1 AXL AXL UFO, Tyro7(r) Ark(m) NM_001699 NP_001690 19q13.1family MER MERTK, NYK, Eyk(ch) NM_006343 NP_006334 2q14.1 TYRO3 RSE,SKY, BRT, DTK, NM_006293 NP_006284 15q15.1-q21.1 TIF DDR DDR1 CAK, TRKE,NEP NM_013993 NP_001945 6p21.3 family NTRK4, EDDR1, PTK3 DDR2 TKT,TYR010, NTRKR3 NM_006182 NP_006173 1q21-q22 EGFR EGFR ERBB, ERBB1NM_005228 NP_005219 7p12 family ERBB2 HER2, Neu(r), NGL NM_004448NP_004439 17q11.2-q12 ERBB3 HER3 NM_001982 NP_001973 12q13 ERBB4 HER4NM_005235 NP_005226 2q33.3-q34 EPH family EPHA1 EPH, EPHT NM_005232NP_005223 7q32-q36 EPHA2 ECK, Sek(m), Myk2(m) NM_004431 NP_004422 1p34EPHA3 HEK, ETK1, Tyro4(r), NM_005233 NP_005224 3p11.2 Mek4(m), Cek4(ch)EPHA4 HEK8, Tyro1(r), Sek1(m), NM_004438 NP_004429 2q36qter Cek8(ch)EPHA5 HEK7, Ehk(r), Bsk(r), L36644 P54756 Cek7(ch) EPHA6 DKFZp434C1418,Ehk2(r) AL133666 EPHA7 HEK11, Mdk1(m), NM_004440 NP_004431 6q21 Ebk(m),Ehk3(r), Cek11(ch) EPHA8 HEK3, KIAA1459, Eek(r), AB040892 CAB816121q23-q24 Cek10(ch) EPHB1 NET, EPHT2, HEK6, NM_004441 NP_004432 3q21-q23Elk(r), Cek6(ch) EPHB2 HEK5, ERK, DRT, AF025304 AAB94602 1p36.1-p35EPHT3, Tyro5(r), Nuk(m), Sek3(m), Cek5(ch) EPHB3 HEK2, Tyro6, Mdk5(m),NM_004443 NP_004434 3q21-qter Sek4(m) EPHB4 HTK, Tyro11(r), NM_004444NP_004435 Mdk2(m), Myk1 (m) EPHB6 HEP, Mep(m), Cek1(ch) NM_004445NP_004436 7q33-q35 FGFR FGFR1 FLT2, bFGFR, FLG, N- M34641 AAA358358p11.2 family SAM FGFR2 KGFR, K-SAM, Bek(m), NM_000141 NP_000132 10q26CFD1, JWS, Cek3(ch) FGFR3 HBGFR, ACH, Cek2(ch) NM_000142 NP_0001334p16.3 FGFR4 NM_002011 NP_002002 5q35.1-qter INSR IGF1R JTK13 NM_000875NP_000866 15q25-q26 family INSR IR NM_000208 NP_000199 19p13.3-p13.2INSRR IRR J05046 AAC31759 1q21-q23 MET MET HGFR NM_000245 NP_000236 7q31Family RON MST1R, CDw136, NM_002447 NP_002438 3p21.3 Fv2(m), STK(m),SEA(ch) MUSK MUSK Nsk2(m), Mlk1(m), NM_005592 NP_005583 9q31.3-q32family Mk2(m) PDGFR CSF1R FMS, C-FMS, CD115 NM_005211 NP_005202 5q31-q32family FLT3 FLK2, STK1, CD135 NM_004119 NP_0041110 13q12 KIT Sfr(m),CKIT NM_000222 NP_000213 4q11-q12 PDGFRA NM_006206 NP_006197 4q11-q13PDGFRB PDGFR, JTK12 NM_002609 NP_002600 5q31-q32 PTK7 PTK7 CCK4, KLG(ch)NM_002821 NP_002812 6p21.2-p12.2 family RET family RET MEN2A/B, HSCR1,X12949 P07949 10q11.2 MTC1 ROR ROR1 NTRKR1 NM_005012 NP_005003 1p32-p31family ROR2 NTRKR2 NM_004560 NP_004551 ROS family ROS1 MCF3 NM_002944NP_002935 6q22 RYK RYK Vlk(m), Mrk(m) S59184 AAB263411 3q22 family TIEfamily TEK TIE2 NM_000459 NP_000450 9p21 TIE TIE1, JTK14 NM_005424NP_005415 1p34-p33 TRK family NTRK1 TRK, TRKA NM_002529 NP_0025201q21-q22 NTRK2 TRKB NM_006180 NP_006171 9q22.1 NTRK3 TRKC NM_002530NP_002521 15q25 VEGFR VEGFR1 FLT1 NM_002019 NP_002010 13q12 familyVEGFR2 KDR, FLK1 AAB88005 4q11-q12 VEGFR3 FLT4, PCL NM_002020 NP_0020115q34-q35 AATYK AATYK AATK, KIAA0641 NM_004920 NP_004911 17q25.3 familyAATYK2 KIAA1079 NM_014916 NP_055731 7q21-q22 AATYK3 19q13.2-q13.3

There are numerous advantages to the prime editing-CID system. One suchadvantage is that it can replace endogenous ligands, which are typicallyproteins that pose complications in manufacturing, cost, delivery,production, or storage, with drug-like small-molecules that can beorally administered instead of administered by IV or injection, arereadily prepared from FDA-approved drugs (or are themselves alreadydrugs), and do not incur special production or storage costs typicallyassociated with protein drugs. Another advantage is that the edit aloneshould have no physiological effect. The amount of target proteindimerization can be controlled by dosing the small-molecule CID.Further, target protein dimerization is readily and rapidly reversibleby adding the monomeric form of the CID. Yet another advantage is thatin instances where a single ligand targets multiple receptors,selectivity can be achieved by prime-editing only one receptor. Finally,depending on the delivery method used for prime editing, it may also bepossible to restrict editing to a localized tissue or organ, allowingfor inducible receptor activation only in specific areas.

If editing efficiencies are high enough with prime editing that twoseparate editing events could occur at high levels, it would also bepossible to tag two proteins of interest with different small-moleculebinding domains (such as FKBP and cyclophilin) and induceheterodimerizations with small molecule heterodimers (such as anFK506-cyclosporin A dimer).

The fusion of FKBP12 or other small-molecule binding proteins to nativeproteins has been accomplished, generally by overexpression from plasmidin tissue culture. Subsequent chemical-induced dimerization has beendemonstrated to induce phenotypic changes to cells producing the fusionproteins.

The following references are cited above in the Section G and areincorporated herein by reference.

-   1. Crabtree, G. R. & Schreiber, S. L. Three-part inventions:    intracellular signaling and induced proximity. Trends Biochem. Sci.    21, 418-22 (1996).-   2. Liu, J. et al. Calcineurin Is a Common Target of A and FKBP-FK506    Complexes. Cell 66, 807-815 (1991).-   3. Keith, C. T. et al. A mammalian protein targeted by Gi-arresting    rapamycin-receptor complex. Nature 369, 756-758 (2003).-   4. Spencer, D. M., Wandless, T. J., Schreiber, S. L. S. &    Crabtree, G. R. Controlling signal transduction with synthetic    ligands. Science 262, 1019-24 (1993).-   5. Pruschy, M. N. et al. Mechanistic studies of a signaling pathway    activated by the organic dimerizer FK1012. Chem. Biol. 1, 163-172    (1994).-   6. Spencer, D. M. et al. Functional analysis of Fas signaling in    vivo using synthetic inducers of dimerization. Curr. Biol. 6,    839-847 (1996).-   7. Belshaw, P. J., Spencer, D. M., Crabtree, G. R. &    Schreiber, S. L. Controlling programmed cell death with a    cyclophilin-cyclosporin-based chemical inducer of dimerization.    Chem. Biol. 3, 731-738 (1996).-   8. Yang, J. X., Symes, K., Mercola, M. & Schreiber, S. L.    Small-molecule control of insulin and PDGF receptor signaling and    the role of membrane attachment. Curr. Biol. 8, 11-18 (1998).-   9. Belshaw, P. J., Ho, S. N., Crabtree, G. R. & Schreiber, S. L.    Controlling protein association and subcellular localization with a    synthetic ligand that induces heterodimerization of proteins. Proc.    Natl. Acad. Sci. 93, 4604-4607 (2002).-   10. Stockwell, B. R. & Schreiber, S. L. Probing the role of    homomeric and heteromeric receptor interactions in TGF-β signaling    using small molecule dimerizers. Curr. Biol. 8, 761-773 (2004).-   11. Spencer, D. M., Graef, I., Austin, D. J., Schreiber, S. L. &    Crabtree, G. R. A general strategy for producing conditional alleles    of Src-like tyrosine kinases. Proc. Natl. Acad. Sci. 92, 9805-9809    (2006).-   12. Holsinger, L. J., Spencer, D. M., Austin, D. J.,    Schreiber, S. L. & Crabtree, G. R. Signal transduction in T    lymphocytes using a conditional allele of Sos. Proc. Natl. Acad.    Sci. 92, 9810-9814 (2006).-   13. Myers, M. G. Insulin Signal Transduction and the IRS Proteins.    Annu. Rev. Pharmacol. Toxicol. 36, 615-658 (1996).-   14. Watowich, S. S. The erythropoietin receptor: Molecular structure    and hematopoietic signaling pathways. J. Investig. Med. 59,    1067-1072 (2011).-   15. Blau, C. A., Peterson, K. R., Drachman, J. G. & Spencer, D. M. A    proliferation switch for genetically modified cells. Proc. Natl.    Acad. Sci. 94, 3076-3081 (2002).-   16. Clackson, T. et al. Redesigning an FKBP-ligand interface to    generate chemical dimerizers with novel specificity. Proc. Natl.    Acad. Sci. 95, 10437-10442 (1998).-   17. Diver, S. T. & Schreiber, S. L. Single-step synthesis of    cell-permeable protein dimerizers that activate signal transduction    and gene expression. J. Am. Chem. Soc. 119, 5106-5109 (1997).-   18. Guo, Z. F., Zhang, R. & Liang, F. Sen. Facile functionalization    of FK506 for biological studies by the thiol-ene ‘click’ reaction.    RSC Adv. 4, 11400-11403 (2014).-   19. Robinson, D. R., Wu, Y.-M. & Lin, S.-F. The protein tyrosine    kinase family of the human genome. Oncogene 19, 5548-5557 (2000).

L. Use of Prime Editing for Cell Data Recording

The prime editors and the resulting genomic modifications can also beused to study and record cellular processes and development. Forexample, the prime editors described herein may be used to record thepresence and duration of a stimulus to a cell by providing to the cell afirst nucleic acid sequence that encodes a fusion protein with a nucleicacid programmable DNA binding protein (napDNAbp) and a reversetranscriptase, and providing the cell at least a second nucleic acidsequence that encodes a PEgRNA. Either the first, the second, or bothnucleic acid sequences are operably linked to inducible promoters thatare responsive to the cell stimulus such that it induces expression ofthe fusion protein and/or the PEgRNA thereby causing the modification ofa target sequence within the cell.

The prime editors described herein can also be used for cellularbarcoding and lineage tracing. For example, by barcoding each cell witha unique genomic barcode, the prime editor can help reveal the celllineage map by allowing the construction of phylogenetic trees based onthe modifications made in one or more target sequence. Starting fromprogenitor cells, the prime editor system can enable building acell-fate map for single cells in a whole organism, which can bedeciphered by analyzing the modifications made in one or more targetsequence. The method for tracing the linage of cells can includeproviding a nucleic acid encoding a fusion protein with a napDNAbp and areverse transcriptase, and providing at least one second nucleic acidencoding a PEgRNA. A unique cellular barcode can be generated using thefusion protein and the PEgRNA to create one or more modifications in oneor more target sequence, thereby allowing the linage of any cell thatarises from the first cell to be traced using the unique cellularbarcode. The use of prime editors for both cell data recording andlinage tracing is further described in Example 13.

The prime editors can do perform both lineage tracing and cellularsignaling recording by modifying genomic target sequences or integratedpre-designed sequences. Prime editors use a synthetic fusion proteincomprising a Cas9 nickase fragment (including but not limited to theSpCas9 H840A variant) and a reverse transcriptase domain, along with anengineered prime editing guide RNA (PEgRNA). Together, these componentstarget a specific genomic sequence or integrated pre-designed sequenceand install a pre-determined edit. Because the PEgRNA specifies both thetarget genomic sequence and the edited outcome, highly specific andcontrolled genome modification can be achieved simultaneously usingmultiple PEgRNA within the same cell. Accessible genome modificationsinclude all single nucleotide substitutions, small- to medium-sizedsequence insertions, and small- to medium-sized deletions. Theversatility of this genome editing technology can enable temporallycoupled, signal-specific recording within cells.

The use of prime editors for cell data recording can includecompositions (e.g., nucleic acids), cells, systems, kits, and methodsfor recording the strength and/or duration of endogenous or exogenousstimuli over the course of a cell's lifetime. The cell data recordingsystem can include a fusion protein consisting of a napDNAbp (e.g., aCas9 domain) and a reverse transcriptase operably linked to a promoterthat induces the expression of the fusion protein to induce changes bycreating targeted and sequence-specified genomic insertions, deletions,or mutations in response to a stimulus or change in the cell. Incontrast to digital memory devices that store information (e.g., thepresence or absence of a stimulus) in one of two distinct states (i.e.,“on” or “off”), these cell data recorders can induce permanent marks incellular DNA in a manner that reflects both the strength (i.e.,amplitude) and duration of one or more stimuli. Thus, in some aspects,cell data recording systems have the ability to simultaneously recordmultiple cell states, including, for example, exposure to a smallmolecule, a protein, a peptide, an amino acid, a metabolite, aninorganic molecule, an organometallic molecule, an organic molecule, adrug or drug candidate, a sugar, a lipid, a metal, a nucleic acid, amolecule produced during the activation of an endogenous or an exogenoussignaling cascade, light, heat, sound, pressure, mechanical stress,shear stress, or a virus or other microorganism. These cell datarecorders can employ sequencing technologies (e.g., high-throughputsequencing) to measure readout (e.g., changes in cellular DNA) and arenot dependent on large cell populations for both the recording of astimulus or the readout of the change(s) in cellular DNA induced by thestimulus.

In general, the cell data recorder systems provided herein for use in acell comprise a fusion protein consisting of a napDNAbp and a reversetranscriptase, wherein the nucleic acid sequence encoding the fusionplasmid is operably linked to a promoter (e.g., an inducible promoter ora constitutive promoter). When a stimulus is present, or a change incell state occurs, the stimulus induces the expression of the fusionprotein. Also present within the cell are one or more nucleic acidsencoding at least one PEgRNA that associate with the napDNAbp anddirects the napDNAbp or the fusion protein to a target sequence (i.e.,the PEgRNA is complementary to a target sequence). The nucleic acidencoding PEgRNA may also be, or may alternatively be, operably linked toa promoter (e.g., an inducible promoter or a constitutive promoter).Under the correct stimulus, or correct set of stimuli, both the fusionprotein and the PEgRNA are expressed in the cell, and the PEgRNAassociates with the fusion protein to direct it to a target sequence.This target sequence records the activity of the prime editor, therebyrecording the presence of a stimulus, or a set of stimuli, or a changein cell state. More than one PEgRNA sequence can also be present in thecell, and these additional PEgRNA sequences, which can direct the fusionprotein to distinct target sequences, can each be operably linked to apromoter that senses the presence of a different stimulus, allowingcomplex cell data recorder systems to be constructed for the orderedrecording of the presence and duration of a stimulus, or set of stimuli.In some cases, one or more of the components of the cell data recordersystem (e.g., fusion protein and PEgRNA) may be constitutively expressedin the cell. Exemplary components of the cell data recorder system foruse with the compositions are described herein. Additional suitablecombinations of components provided herein will be apparent to a personof ordinary skill in the art based on this disclosure and knowledge inthe field, and thus are embraced by the scope of this disclosure.

Repeated modification of a DNA target that can be sequenced by targetedamplicon sequencing and/or RNA sequencing (which is particular value forsingle cell recording experiments) can be used to record a host ofimportant biological processes, including activation of signalingcascades, metabolic states, and cellular differentiation programs.Connecting internal and external cellular signals to sequencemodification in the genome is possible for any signal for which a signalresponsive promotor exists. In some embodiments, the promoter is apromoter suitable for use in a prokaryotic system (i.e., a bacterialpromoter). In some embodiments, the promoter is a promoter suitable foruse in a eukaryotic system (i.e., a eukaryotic promoter). In someembodiments, the promoter is a promoter suitable for use in a mammalian(e.g., human) system (i.e., a mammalian promoter). In some embodiments,the promoter is induced by a stimulus (i.e., an inducible promoter). Insome embodiments, the stimulus is a small molecule, a protein, apeptide, an amino acid, a metabolite, an inorganic molecule, anorganometallic molecule, an organic molecule, a drug or drug candidate,a sugar, a lipid, a metal, a nucleic acid, a molecule produced duringthe activation of an endogenous or an exogenous signaling cascade,light, heat, sound, pressure, mechanical stress, shear stress, or avirus or other microorganism, change in pH, or change inoxidation/reduction state. In some embodiments, the stimulus is a light.In some embodiments, the stimulus is a virus. In some embodiments, thestimulus is a small molecule. In some embodiments, the stimulus is anantibiotic. In some embodiments, the stimulus is anhydrotetracycline ordoxycycline. In some embodiments, the stimulus is a sugar. In someembodiments, the stimulus is arabinose, rhamnose, or IPTG. In someembodiments, the stimulus is a signaling molecule produced during anactivated signaling cascade (e.g., beta-catenin produced during anactivated Wnt signaling cascade). Additional promoters that detectsignaling molecules can be generated to induce the expression of thenucleic acid sequence operably linked to the promoter, for example,promoters that record an endogenous pathway, including immune response(IL-2 promoter), a cAMP responsive element (CREB), NFκB signaling,interferon response, P53 (DNA damage), Sox2, TGF-ß signaling (SMAD), Erk(e.g., from an activated Ras/Raf/Mek/Erk cascade), PI3K/AKT (e.g., froman activated Ras/PI3K/Akt cascade), heat shock, Notch signaling, Oct4,an aryl hydrocarbon receptor, or an AP-1 transcription factor. In someembodiments, the promoter is a constitutive promoter. In someembodiments, the promoter is a promoter listed in Table 3. Additionalsuitable promoters for use in both prokaryotic and eukaryotic systemswill be apparent to those of ordinary skill in the art based on thisdisclosure and knowledge in the field, and are within the scope of thepresent disclosure.

Prime editors can also be used to trace cellular lineages. Repeatedsequence modifications can be used to generate unique cellular barcodesto track individual cells. The arrays of barcodes, their order, and sizecan all be used to infer cellular lineages. For example, the insertionof homology sequences (i.e., sequences 3′ of the Cas9 nick location),and in particular homology sequences with associated barcodes, appear tobe particularly useful lineage prime editor strategies. These systemscan be designed such that successive rounds of editing result in theinsertion of a barcode from a PEgRNA cassette that cannot be modified byother PEgRNA editing events in the same cell. The barcoding system canutilize multiple barcodes that can be associated with a given stimulus.This system can preserve the majority of the target protospacers butalter the seed sequence, PAM, and downstream adjacent nucleotides. Thisenables multiple signals to be connected to one editing locus withoutsignificant re-designing of the PEgRNAs being used. The strategy canenable multiplexed barcode insertions in response to a large number ofcellular stimuli (either internal or external) at a single locus. Itcould enable the recording of intensity, duration, and order of as manysignals as there exist unique barcodes (which can be designed withmultiple N nucleotides to generate 4{circumflex over ( )}N possiblebarcodes, for example a 5-nt barcode would enable recording of4{circumflex over ( )}5 or 1024 unique signals at once). This system canbe used both in vitro and in vivo.

M. Use of Prime Editing to Modulate Biomolecule Activity

The use of prime editors described herein may also be used to regulatethe subcellular localization and modification states of biomolecules,such as DNA, RNA, and proteins. Specific biological functions, liketranscriptional control, cellular metabolism, and signal transductioncascades, are carefully orchestrated in particular locations within thecell. The ability to traffic proteins to these and other unique cellularcompartments could provide an opportunity to alter a number ofbiological processes.

Accordingly, prime editing can be used to install genetically encodedhandles that will allow for altered modification states and thesubcellular trafficking of biomolecules with a genetically encodedsignal (e.g. proteins, lipids, sugars, and nucleic acids). In variousembodiments, the target biomolecules for prime editor-mediatedmedication are DNA. For example, DNA could be modified by installing anumber of DNA sequences that change the accessibility of the targetlocus, which could lead to either increased or decreased transcriptionof desired sequences. In other embodiments, the target biomolecules forprime editor-mediated medication are RNA. For example, the activity ofRNA can be modified by changing its cellular localization, interactingpartners, structural dynamics, or thermodynamics of folding. In yetother embodiments, the target biomolecules for prime editor-mediatedmedication are protein. Proteins can be modified to impactpost-translational modifications, protein motifs can be installed tochange the subcellular localization of the protein, or proteins can bemodified to either create or destroy their ability to exist withinprotein-protein complexation events.

This application of prime editing can be further described in Example14.

DNA Modifications

One target biomolecule for PE-mediated modification is DNA.Modifications to DNA could be made to install a number of DNA sequencesthat change the accessibility of the target locus. Chromatinaccessibility controls gene transcriptional output. Installation ofmarks to recruit chromatin compacting enzymes should decrease thetranscriptional output of neighboring genes, while installation ofsequences associated with chromatin opening should make regions moreaccessible and in turn increase transcription. Installation of morecomplex sequence motifs that mirror native regulatory sequences shouldprovide more nuanced and biologically sensitive control than thecurrently available dCas9 fusions to different epigenetic reader,writer, or eraser enzymes-tools that typically install large numbers ofa single type of mark that may not have a particular biologicalantecedent. Installation of sequences that will bring two loci intoclose proximity, or bring loci into contact with the nuclear membrane,should also alter the transcriptional output of those loci as has beendemonstrated in the burgeoning field of 3-D genomic architecture.

RNA Modifications

Modifications to RNAs can also be made to alter their activity bychanging their cellular localization, interacting partners, structuraldynamics, or thermodynamics of folding. Installation of motifs that willcause translational pausing or frameshifting could change the abundanceof mRNA species through various mRNA processing mechanisms. Modifyingconsensus splice sequences would also alter the abundance and prevalenceof different RNA species. Changing the relative ratio of differentsplice isoforms would predictably lead to a change in the ratio ofprotein translation products, and this could be used to alter manybiological pathways. For instance, shifting the balance of mitochondrialversus nuclear DNA repair proteins would alter the resilience ofdifferent cancers to chemotherapeutic reagents. Furthermore, RNAs couldbe modified with sequences that enable binding to novel protein targets.A number of RNA aptamers have been developed that bind with highaffinity to cellular proteins. Installation of one of these aptamerscould be used to either sequester different RNA species through bindingto a protein target that will prevent their translation, biologicalactivity, or to bring RNA species to specific subcellular compartments.Biomolecule degradation is another class of localization modification.

For example, RNA methylation is used to regulate RNAs within the cell.Consensus motifs for methylation could be introduced into target RNAcoding sequences with PE. RNAs could also be modified to includesequences that direct nonsense mediated decay machinery or other nucleicacid metabolism pathways to degrade the target RNA species would changethe pool of RNAs in a cell. Additionally, RNA species could be modifiedto alter their aggregation state. Sequences could be installed on singleRNAs of interest or multiple RNAs to generate RNA tangles that wouldrender them ineffective substrates for translation or signaling.

Protein Modifications

Modifications to proteins via post-translational modification (PTM) alsorepresent an important class of biomolecule manipulation that can becarried out with PE. As with RNA species, changing the abundance ofproteins in a cell is an important capability of PE. Editing can be doneto install stop codons in an open reading frame—this will eliminatefull-length product from being produced by the edited DNA sequence.Alternatively, peptide motifs can be installed that cause the rate ofprotein degradation to be altered for a target protein. Installation ofdegradation tags into a gene body could be used to alter the abundanceof a protein in a cell. Moreover, introduction of degrons that areinduced by small molecules could enable temporal control over proteindegradation. This could have important implications for both researchand therapeutics as researchers could readily assess whether smallmolecule-mediated therapeutic protein degradation of a given target wasa viable therapeutic strategy. Protein motifs could also be installed tochange the subcellular localization of a protein. Amino acid motifs canbe installed to preferentially traffic proteins to a number ofsubcellular compartments including the nucleus, mitochondria, cellmembrane, peroxisome, lysosome, proteasome, exosome, and others.

Installing or destroying motifs modified by PTM machinery can alterprotein post-translational modifications. Phosphorylation,ubiquitylation, glycosylation, lipidation (e.g. farnesylation,myristoylation, palmitoylation, prenylation, GPI anchors),hydroxylation, methylation, acetylation, crotonylation, SUMOylation,disulfide bond formations, side chain bond cleavage events, polypeptidebackbone cleavage events (proteolysis), and a number of other proteinPTMs have been identified. These PTMs change protein function, often bychanging subcellular localization. Indeed, kinases often activatedownstream signaling cascades via phosphorylation events. Removal of thetarget phosphosite would prevent signal transduction. The ability tosite-specifically ablate or install any PTM motif while retainingfull-length protein expression would be an important advance for bothbasic research and therapeutics. The sequence installation scope andtarget window of PE make it well suited for broad PTM modificationspace.

Removal of lipidation sites should prevent the trafficking of proteinsto cell membranes. A major limitation to current therapeutics thattarget post-translational modification processes is their specificity.Farnesyl transferase inhibitors have been tested extensively for theirability to eliminate KRAS localization at cell membranes. Unfortunately,global inhibition of farnesylation comes with numerous off targeteffects that have prevented broad use of these small molecules.Similarly, specific inhibition of protein kinases with small moleculescan be very challenging due to the large size of the human genome andsimilarities between various kinases. PE offers a potential solution tothis specificity problem, as it enables inhibition of modification ofthe target protein by ablation of the modification site instead ofglobal enzyme inhibition. For example, removal of the lapidated peptidemotif in KRAS would be a targeted approach that could be used in placeof farnesyl transferase inhibition. This approach is the functionalinverse of inhibiting a target protein activity by installing alipid-targeting motif on a protein not designed to be membrane bound.

PE can also be used to instigate protein-protein complexation events.Proteins often function within complexes to execute their biologicalactivity. PE can be used to either create or destroy the ability ofproteins to exist within these complexes. To eliminate complex formationevents, amino acid substitutions or insertions along the protein:protein interface could be installed to disfavor complexation. SSX18 isa protein component of the BAF complex, an important histone-remodelingcomplex. Mutations in SSX18 drive synovial sarcomas. PE could be used toinstall side chains that prevent SSX18 from binding to its proteinpartners in the complex to prevent its oncogenic activity. PE could alsobe used to remove the pathogenic mutations to restore WT activity ofthis protein. Alternatively, PE could be used to keep proteins withineither their native complex or to drag them to participate ininteractions that are unrelated to their native activity to inhibittheir activity. Forming complexes that maintain one interaction stateover another could represent an important therapeutic modality. Alteringprotein: protein interfaces to decrease the Kd of the interaction wouldkeep those proteins stuck to one another longer. As protein complexescan have multiple signaling complexes, like n-myc driving neuroblastomasignaling cascades in disease but otherwise participating in healthytranscriptional control in other cells. PE could be used to installmutations that drive n-myc association with healthy interactionspartners and decrease its affinity for oncogenic interaction partners.

REFERENCES CITED IN SECTION I

Each of the following references are incorporated herein by reference.

-   1. Selective Target Protein Degradation via Phthalimide Conjugation.    Winter et al. Science. Author manuscript; available in PMC 2016 Jul.    8.-   2. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX    oncogenic fusion in synovial sarcoma. Kadoch and Crabtree. Cell.    2013 Mar. 28; 153(1):71-85. doi: 10.1016/j.cell.2013.02.036.-   3. Ribosomal frameshifting and transcriptional slippage: From    genetic steganography and cryptography to adventitious use. Atkins    et al. Nucleic Acids Research, Volume 44, Issue 15, 6 Sep. 2016,    Pages 7007-7078.-   4. Transcriptional Regulation and its Misregulation in Disease. Lee    and Young. Cell. Author manuscript; available in PMC 2014 Mar. 14.-   5. Protein localization in disease and therapy. Mien-Chie Hung,    Wolfgang Link Journal of Cell Science 2011 124: 3381-3392.-   6. Loss of post-translational modification sites in disease. Li et    al. Pac Symp Biocomput. 2010:337-47. PTMD: A Database of Human    Disease-associated Post-translational Modifications. Xu et al.    Genomics Proteomics Bioinformatics. 2018 August; 16(4):244-251. Epub    2018 Sep. 21.-   7. Post-transcriptional gene regulation by mRNA modifications. Zhao    et al. Nature Reviews Molecular Cell Biology volume 18, pages 31-42    (2017).

N. Improved Design Aspects of PEgRNAs for Prime Editing

In other embodiments, the prime editing system may include the use ofPEgRNA designs and strategies that can improve prime editing efficiency.These strategies seek to overcome some issues that exist because of themulti-step process required for prime editing. For example, unfavorableRNA structures that can form within the PEgRNA can result in theinhibition of DNA edits being copied from the PEgRNA into the genomiclocus. These limitations could be overcome through the redesign andengineering of the PEgRNA component. These redesigns could improve primeeditor efficiency, and could allow the installation of longer insertedsequences into the genome.

Accordingly, in various embodiments, the PEgRNA designs can result inlonger PEgRNAs by enabling efficient expression of functional PEgRNAsfrom non-polymerase III (pol III) promoters, which would avoid the needfor burdensome sequence requirements. In other embodiments, the core,Cas9-binding PEgRNA scaffold can be improved to improve efficacy of thesystem. In yet other embodiments, modifications can be made to thePEgRNA to improve reverse transcriptase (RT) processivity, which wouldenable the insertion of longer sequences at the targeted genomic loci.In other embodiments, RNA motifs can be added to the 5′ and/or 3′termini of the PEgRNA to improve stability, enhance RT processivity,prevent misfolding of the PEgRNA, and/or recruit additional factorsimportant for genome editing. In yet another embodiment, a platform isprovided for the evolution of PEgRNAs for a given sequence target thatcould improve the PEgRNA scaffold and enhance prime editor efficiency.These designs could be used to improve any PEgRNA recognized by any Cas9or evolved variant thereof.

This application of prime editing can be further described in Example15.

The PEgRNAs may include additional design improvements that may modifythe properties and/or characteristics of PEgRNAs thereby improving theefficacy of prime editing. In various embodiments, these improvementsmay belong to one or more of a number of different categories, includingbut not limited to: (1) designs to enable efficient expression offunctional PEgRNAs from non-polymerase III (pol III) promoters, whichwould enable the expression of longer PEgRNAs without burdensomesequence requirements; (2) improvements to the core, Cas9-binding PEgRNAscaffold, which could improve efficacy; (3) modifications to the PEgRNAto improve RT processivity, enabling the insertion of longer sequencesat targeted genomic loci; and (4) addition of RNA motifs to the 5′ or 3′termini of the PEgRNA that improve PEgRNA stability, enhance RTprocessivity, prevent misfolding of the PEgRNA, or recruit additionalfactors important for genome editing.

In one embodiment, PEgRNA could be designed with polII promoters toimprove the expression of longer-length PEgRNA with larger extensionarms. sgRNAs are typically expressed from the U6 snRNA promoter. Thispromoter recruits pol III to express the associated RNA and is usefulfor expression of short RNAs that are retained within the nucleus.However, pol III is not highly processive and is unable to express RNAslonger than a few hundred nucleotides in length at the levels requiredfor efficient genome editing. Additionally, pol III can stall orterminate at stretches of U's, potentially limiting the sequencediversity that could be inserted using a PEgRNA. Other promoters thatrecruit polymerase II (such as pCMV) or polymerase I (such as the U1snRNA promoter) have been examined for their ability to express longersgRNAs. However, these promoters are typically partially transcribed,which would result in extra sequence 5′ of the spacer in the expressedPEgRNA, which has been shown to result in markedly reduced Cas9:sgRNAactivity in a site-dependent manner. Additionally, while polII-transcribed PEgRNAs can simply terminate in a run of 6-7 U's, PEgRNAstranscribed from pol II or pol I would require a different terminationsignal. Often such signals also result in polyadenylation, which wouldresult in undesired transport of the PEgRNA from the nucleus. Similarly,RNAs expressed from pol II promoters such as pCMV are typically5′-capped, also resulting in their nuclear export.

Previously, Rinn and coworkers screened a variety of expressionplatforms for the production of long-noncoding RNA-(lncRNA) taggedsgRNAs¹⁸³. These platforms include RNAs expressed from pCMV and thatterminate in the ENE element from the MALAT1 ncRNA from humans¹⁸⁴ thePAN ENE element from KSHV¹⁸⁵, or the 3′ box from U1 snRNA¹⁸⁶. Notably,the MALAT1 ncRNA and PAN ENEs form triple helices protecting thepolyA-tail^(184, 187). These constructs could also enhance RNAstability. It is contemplated that these expression systems will alsoenable the expression of longer PEgRNAs.

In addition, a series of methods have been designed for the cleavage ofthe portion of the pol II promoter that would be transcribed as part ofthe PEgRNA, adding either a self-cleaving ribozyme such as thehammerhead¹⁸⁸, pistol¹⁸⁹, hatchet¹⁸⁹, hairpin¹⁹⁰, VS¹⁹¹, twister¹⁹², ortwister sister¹⁹² ribozymes, or other self-cleaving elements to processthe transcribed guide, or a hairpin that is recognized by Csy4¹⁹³ andalso leads to processing of the guide. Also, it is hypothesized thatincorporation of multiple ENE motifs could lead to improved PEgRNAexpression and stability, as previously demonstrated for the KSHV PANRNA and element¹⁸⁵. It is also anticipated that circularizing the PEgRNAin the form of a circular intronic RNA (ciRNA) could also lead toenhanced RNA expression and stability, as well as nuclearlocalization¹⁹⁴.

In various embodiments, the PEgRNA may include various above elements,as exemplified by the following sequence.

Non-limiting example 1 - PEgRNA expression platformconsisting of pCMV, Csy4 hairpin, the PEgRNA, and  MALAT1 ENE(SEQ ID NO: 501) TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGATGCTGGTGGTTGGCACTCCTGGTTTCCAGGACGGGGTTCAAATCCCTGCGGCGTCTTTGCTTTGACTNon-limiting example 2 - PEgRNA expression platformconsisting of pCMV, Csy4 hairing, the PEgRNA, and  PAN ENE(SEQ ID NO: 502) TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAANon-limiting example 3 - PEgRNA expression platformconsisting of pCMV, Csy4 hairing, the PEgRNA, and  3xPAN ENE(SEQ ID NO: 503) TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAAACACACTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAATCTCTCTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAANon-limiting example 4 - PEgRNA expression platformconsisting of pCMV, Csy4 hairing, the PEgRNA, and  3′ box (SEQ ID NO: 504) TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAANon-limiting example 5 - PEgRNA expression platformconsisting of pU1, Csy4 hairping, the PEgRNA, and  3′ box(SEQ ID NO: 505) CTAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGGAAAAAGGGAGAGGCAGACGTCACTTCCCCTTGGCGGCTCTGGCAGCAGATTGGTCGGTTGAGTGGCAGAAAGGCAGACGGGGACTGGGCAAGGCACTGTCGGTGACATCACGGACAGGGCGACTTCTATGTAGATGAGGCAGCGCAGAGGCTGCTGCTTCGCCACTTGCTGCTTCACCACGAAGGAGTTCCCGTGCCCTGGGAGCGGGTTCAGGACCGCTGATCGGAAGTGAGAATCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCGGGAGTGCGCGGGGCAAGTGACCGTGTGTGTAAAGAGTGAGGCGTATGAGGCTGTGTCGGGGCAGAGGCCCAAGATCTCAGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTCAGCAAGTTCAGAGAAATCTGAACTTGCTGGATTTTTGGAGCAGGGAGATGGAATAGGAGCTTGCTCCGTCCACTCCACGCATCGACCTGGTATTGCAGTACCTCCAGGAACGGTGCACCCACTTTCTGGAGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAA

In various other embodiments, the PEgRNA may be improved by introducingimprovements to the scaffold or core sequences. This can be done byintroducing known

The core, Cas9-binding PEgRNA scaffold can likely be improved to enhancePE activity. Several such approaches have already been demonstrated. Forinstance, the first pairing element of the scaffold (P1) contains aGTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such runs of Ts have beenshown to result in pol III pausing and premature termination of the RNAtranscript. Rational mutation of one of the T-A pairs to a G-C pair inthis portion of P1 has been shown to enhance sgRNA activity, suggestingthis approach would also be feasible for PEgRNAs¹⁹⁵. Additionally,increasing the length of P1 has also been shown to enhance sgRNA foldingand lead to improved activity¹⁹⁵, suggesting it as another avenue forthe improvement of PEgRNA activity. Example improvements to the core caninclude:

PEgRNA containing a 6 nt extension to P1 (SEQ ID NO: 228)GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTTPEgRNA containing a T-A to G-C mutation within P1 (SEQ ID NO: 229)GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT

In various other embodiments, the PEgRNA may be improved by introducingmodifications to the edit template region. As the size of the insertiontemplated by the PEgRNA increases, it is more likely to be degraded byendonucleases, undergo spontaneous hydrolysis, or fold into secondarystructures unable to be reverse-transcribed by the RT or that disruptfolding of the PEgRNA scaffold and subsequent Cas9-RT binding.Accordingly, it is likely that modification to the template of thePEgRNA might be necessary to affect large insertions, such as theinsertion of whole genes. Some strategies to do so include theincorporation of modified nucleotides within a synthetic orsemi-synthetic PEgRNA that render the RNA more resistant to degradationor hydrolysis or less likely to adopt inhibitory secondarystructures¹⁹⁶. Such modifications could include 8-aza-7-deazaguanosine,which would reduce RNA secondary structure in G-rich sequences;locked-nucleic acids (LNA) that reduce degradation and enhance certainkinds of RNA secondary structure; 2′-O-methyl, 2′-fluoro, or2′-O-methoxyethoxy modifications that enhance RNA stability. Suchmodifications could also be included elsewhere in the PEgRNA to enhancestability and activity. Alternatively or additionally, the template ofthe PEgRNA could be designed such that it both encodes for a desiredprotein product and is also more likely to adopt simple secondarystructures that are able to be unfolded by the RT. Such simplestructures would act as a thermodynamic sink, making it less likely thatmore complicated structures that would prevent reverse transcriptionwould occur. Finally, one could also split the template into two,separate PEgRNAs. In such a design, a PE would be used to initiatetranscription and also recruit a separate template RNA to the targetedsite via an RNA-binding protein fused to Cas9 or an RNA recognitionelement on the PEgRNA itself such as the MS2 aptamer. The RT couldeither directly bind to this separate template RNA, or initiate reversetranscription on the original PEgRNA before swapping to the secondtemplate. Such an approach could enable long insertions by bothpreventing misfolding of the PEgRNA upon addition of the long templateand also by not requiring dissociation of Cas9 from the genome for longinsertions to occur, which could possibly be inhibiting PE-based longinsertions.

In still other embodiments, the PEgRNA may be improved by introducingadditional RNA motifs at the 5′ and 3′ termini of the PEgRNAs. Severalsuch motifs—such as the PAN ENE from KSHV and the ENE from MALAT1 werediscussed above as possible means to terminate expression of longerPEgRNAs from non-pol III promoters. These elements form RNA triplehelices that engulf the polyA tail, resulting in their being retainedwithin the nucleus^(184,187). However, by forming complex structures atthe 3′ terminus of the PEgRNA that occlude the terminal nucleotide,these structures would also likely help prevent exonuclease-mediateddegradation of PEgRNAs.

Other structural elements inserted at the 3′ terminus could also enhanceRNA stability, albeit without enabling termination from non-pol IIIpromoters. Such motifs could include hairpins or RNA quadruplexes thatwould occlude the 3′ terminus¹⁹⁷, or self-cleaving ribozymes such as HDVthat would result in the formation of a 2′-3′-cyclic phosphate at the 3′terminus and also potentially render the PEgRNA less likely to bedegraded by exonucleases¹⁹⁸. Inducing the PEgRNA to cyclize viaincomplete splicing—to form a ciRNA—could also increase PEgRNA stabilityand result in the PEgRNA being retained within the nucleus¹⁹⁴.

Additional RNA motifs could also improve RT processivity or enhancePEgRNA activity by enhancing RT binding to the DNA-RNA duplex. Additionof the native sequence bound by the RT in its cognate retroviral genomecould enhance RT activity¹⁹⁹. This could include the native primerbinding site (PBS), polypurine tract (PPT), or kissing loops involved inretroviral genome dimerization and initiation of transcription¹⁹⁹.

Addition of dimerization motifs—such as kissing loops or a GNRAtetraloop/tetraloop receptor pair²⁰⁰—at the 5′ and 3′ termini of thePEgRNA could also result in effective circularization of the PEgRNA,improving stability. Additionally, it is envisioned that addition ofthese motifs could enable the physical separation of the PEgRNA spacerand primer, prevention occlusion of the spacer which would hinder PEactivity. Short 5′ or 3′ extensions to the PEgRNA that form a smalltoehold hairpin in the spacer region could also compete favorablyagainst the annealing region of the PEgRNA binding the spacer. Finally,kissing loops could also be used to recruit other template RNAs to thegenomic site and enable swapping of RT activity from one RNA to theother. Example improvements include, but are not limited to:

PEgRNA-HDV fusion (SEQ ID NO: 230)GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT PEgRNA-MMLV kissing loop(SEQ ID NO: 231) GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGA GACGTCCCACCTTTTTTTPEgRNA-VS ribozyme kissing loop (SEQ ID NO: 232)GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTCCATCAGTTGACACCCTGAGGTTTTTTT PEgRNA-GNRA tetraloop/tetraloop receptor(SEQ ID NO: 233) GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTUACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTTPEgRNA template switching secondary RNA-HDV fusion (SEQ ID NO: 234)TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT

PEgRNA scaffold could be further improved via directed evolution, in ananalogous fashion to how SpCas9 and prime editor (PE) have beenimproved. Directed evolution could enhance PEgRNA recognition by Cas9 orevolved Cas9 variants. Additionally, it is likely that different PEgRNAscaffold sequences would be optimal at different genomic loci, eitherenhancing PE activity at the site in question, reducing off-targetactivities, or both. Finally, evolution of PEgRNA scaffolds to whichother RNA motifs have been added would almost certainly improve theactivity of the fused PEgRNA relative to the unevolved, fusion RNA. Forinstance, evolution of allosteric ribozymes composed of c-di-GMP-Iaptamers and hammerhead ribozymes led to dramatically improvedactivity²⁰², suggesting that evolution would improve the activity ofhammerhead-PEgRNA fusions as well. In addition, while Cas9 currentlydoes not generally tolerate 5′ extension of the sgRNA, directedevolution will likely generate enabling mutations that mitigate thisintolerance, allowing additional RNA motifs to be utilized.

The present disclosure contemplates any such ways to further improve theefficacy of the prime editing systems disclosed here.

O. Use of Prime Editing with Expanded Targeting Scope

Prime editing (PE) using Streptococcus pyogenes Cas9 (SpCas9) canefficiently install all single base substitutions, insertions,deletions, and combinations thereof at genomic loci where there is asuitably-placed NGG protospacer adjacent motif (PAM) that SpCas9 canefficiently bind. However, in another aspect the methods describedherein broaden the targeting capability of PE by expanding theaccessible PAMs and, therefore, the targetable genomic loci accessiblefor efficient PE. Prime editors using RNA-guided DNA binding proteinsother than SpCas9 enable an expanded targetable scope of genomic loci byallowing access to different PAMs. In addition, use of RNA-guided DNAbinding proteins smaller than SpCas9 also allows for more efficientviral delivery. PE with Cas proteins or other RNA-guided DNA bindingproteins beyond SpCas9 will allow for high efficiency therapeutic editsthat were either inaccessible or inefficient using SpCas9-based PE.

This is expected to be used in situations where SpCas9-based PE iseither inefficient due to non-ideal spacing of an edit to relative to anNGG PAM or the overall size of the SpCas9-based construct is prohibitivefor cellular expression and/or delivery. Specific disease-relevant locisuch as the Huntingtin gene, which has few and poorly located NGG PAMsfor SpCas9 near the target region, can easily be targeted usingdifferent Cas proteins in the PE system such as SpCas9-VRQR whichrecognizes an NGA PAM. Smaller Cas proteins will be used to generatesmaller PE constructs that can be packaged into AAV vectors moreefficiently, enabling better delivery to target tissues. FIG. 61 showsthe reduction to practice of prime editing using Staphylococcus aureusCRISPR-Cas as the RNA-guided DNA binding protein. NT is untreatedcontrol.

FIGS. 62A-62B provide a demonstration of the importance of theprotospacer for efficient installation of a desired edit at a preciselocation with prime editing. This highlights the importance of alternatePAMs and protospacers as novel features of this technology. “n.d.” inFIG. 62A is “not detected.”

FIG. 63 shows the reduction to practice of PE using SpCas9(H840A)-VRQRand SpCas9(H840A)-VRER as the RNA-guided DNA binding protein in a primeeditor system. The SpCas9(H840A)-VRQR napDNAbp is disclosed herein asSEQ ID NO: 87. The SpCas9(H840A)-VRER napDNAbp is disclosed herein asSEQ ID NO: 88. The SpCas9(H840A)-VRER-MMLV RT fusion protein isdisclosed herein as SEQ ID NO: 516, wherein the MMLV RT comprises theD200N, L603W, T330P, T306K, and W313F substitutions relative to the wildtype MMLV RT. The SpCas9(H840A)-VRQR-MMLV RT fusion protein is disclosedherein as SEQ ID NO: 515, wherein the MMLV RT comprises the D200N,L603W, T330P, T306K, and W313F substitutions relative to the wild typeMMLV RT. Seven different loci in the human genome are targeted: 4 withthe SpCas9(H840A)-VRQR-MMLV RT prime editor system and 3 with theSpCas9(H840A)-VRER-MMLV RT system. The amino acid sequences of thetested constructs are as follows:

SACAS9-M-MLV MKRTADGSEFESPKKKRKVGKRNYILGLDIGITSVGYGIIDYETRRT PRIME EDITOR DVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKR KV (SEQ ID NO: 514)SPCAS9(H840A)- MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY VRQR-MALONEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR MURINERRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH LEUKEMIA VIRUSERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL REVERSEAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN TRANSCRIPTASEASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL PRIME EDITORGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 515) SPCAS9(H840A)-MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY VRER-MALONEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR MURINERRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH LEUKEMIA VIRUSERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL REVERSEAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN TRANSCRIPTASEASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL PRIME EDITORGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 516)

As shown in FIG. 63 , the SpCas9(H840A)-VRQR-MMLV RT was operational atPAM sites that included “AGAG” and “GGAG”, with some editing activity at“GGAT” and “AGAT” PAM sequences. The SpCas9(H840A)-VRER-MMLV RT wasoperational at PAM sites that included “AGCG” and “GGCG”, with someediting activity at “TGCG.”

The data demonstrates that prime editing may be conducted usingnapDNAbps which bear different PAM specificities, such as those Cas9variant described herein.

In various embodiments, the napDNAbp (e.g., Cas9) with altered PAMspecificities comprise a combination of mutations that exhibit activityon a target sequence comprising a 5′-NAA-3′ PAM sequence at its 3′-end.In some embodiments, the combination of mutations are present in any oneof the clones listed in Table 1. In some embodiments, the combination ofmutations are conservative mutations of the clones listed in Table 1. Insome embodiments, the Cas9 protein comprises the combination ofmutations of any one of the Cas9 clones listed in Table 1.

TABLE 1 NAA PAM Clones Mutations from wild-type SpCas9 (e.g., SEQ ID NO:18) D177N, K218R, D614N, D1135N, P1137S, E1219V, A1320V, A1323D, R1333KD177N, K218R, D614N, D1135N, E1219V, Q1221H, H1264Y, A1320V, R1333KA10T, I322V, S409I, E427G, G715C, D1135N, E1219V, Q1221H, H1264Y,A1320V, R1333K A367T, K710E, R1114G, D1135N, P1137S, E1219V, Q1221H,H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, R753G, D861N, D1135N,K1188R, E1219V, Q1221H, H1264H, A1320V, R1333K A10T, I322V, S409I,E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G, K1211R, E1219V,Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, V743I, R753G,E762G, D1135N, D1180G, K1211R, E1219V, Q1221H, H1264Y, A1320V, R1333KA10T, I322V, S409I, E427G, R753G, D1135N, D1180G, K1211R, E1219V,Q1221H, H1264Y, S1274R, A1320V, R1333K A10T, I322V, S409I, E427G, A589S,R753G, D1135N, E1219V, Q1221H, H1264H, A1320V, R1333K A10T, I322V,S409I, E427G, R753G, E757K, G865G, D1135N, E1219V, Q1221H, H1264Y,A1320V, R1333K A10T, I322V, S409I, E427G, R654L, R753G, E757K, D1135N,E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, K599R,M631A, R654L, K673E, V743I, R753G, N758H, E762G, D1135N, D1180G, E1219V,Q1221H, Q1256R, H1264Y, A1320V, A1323D, R1333K A10T, I322V, S409I,E427G, R654L, K673E, V743I, R753G, E762G, N869S, N1054D, R1114G, D1135N,D1180G, E1219V, Q1221H, H1264Y, A1320V, A1323D, R1333K A10T, I322V,S409I, E427G, R654L, L727I, V743I, R753G, E762G, R859S, N946D, F1134L,D1135N, D1180G, E1219V, Q1221H, H1264Y, N1317T, A1320V, A1323D, R1333KA10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S,N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H,H1264Y, V1290G, L1318S, A1320V, A1323D, R1333K A10T, I322V, S409I,E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S, Y1016D, G1077D,R1114G, F1134L, D1135N, K1151E, D1180G, E1219V, Q1221H, H1264Y, V1290G,L1318S, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E, V743I,R753G, E762G, N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N,D1180G, E1219V, Q1221H, H1264Y, V1290G, L1318S, A1320V, A1323D, R1333KA10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G,N803S, N869S, L921P, Y1016D, G1077D, F1080S, R1114G, D1135N, D1180G,E1219V, Q1221H, H1264Y, L1318S, A1320V, A1323D, R1333K A10T, I322V,S409I, E427G, E630K, R654L, K673E, V743I, R753G, E762G, Q768H, N803S,N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H,H1264Y, L1318S, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E,F693L, V743I, R753G, E762G, Q768H, N803S, N869S, Y1016D, G1077D, R1114G,F1134L, D1135N, D1180G, E1219V, Q1221H, G1223S, H1264Y, L1318S, A1320V,R1333K A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G,E762G, N803S, N869S, L921P, Y1016D, G1077D, F1801S, R1114G, D1135N,D1180G, E1219V, Q1221H, H1264Y, L1318S, A1320V, A1323D, R1333K A10T,I322V, S409I, E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G,K1211R, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I,E427G, R654L, K673E, V743I, R753G, E762G, M673I, N803S, N869S, G1077D,R1114G, D1135N, V1139A, D1180G, E1219V, Q1221H, A1320V, R1333K A10T,I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,R1114G, D1135N, E1219V, Q1221H, A1320V, R1333K

In some embodiments, the Cas9 protein comprises an amino acid sequencethat is at least 80% identical to the amino acid sequence of a Cas9protein as provided by any one of the variants of Table 1. In someembodiments, the Cas9 protein comprises an amino acid sequence that isat least 85%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence of a Cas9 protein as provided by any one of thevariants of Table 1.

In some embodiments, the Cas9 protein exhibits an increased activity ona target sequence that does not comprise the canonical PAM (5′-NGG-3′)at its 3′ end as compared to Streptococcus pyogenes Cas9 as provided bySEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits anactivity on a target sequence having a 3′ end that is not directlyadjacent to the canonical PAM sequence (5′-NGG-3′) that is at least5-fold increased as compared to the activity of Streptococcus pyogenesCas9 as provided by SEQ ID NO: 18 on the same target sequence. In someembodiments, the Cas9 protein exhibits an activity on a target sequencethat is not directly adjacent to the canonical PAM sequence (5′-NGG-3′)that is at least 10-fold, at least 50-fold, at least 100-fold, at least500-fold, at least 1,000-fold, at least 5,000-fold, at least10,000-fold, at least 50,000-fold, at least 100,000-fold, at least500,000-fold, or at least 1,000,000-fold increased as compared to theactivity of Streptococcus pyogenes as provided by SEQ ID NO: 18 on thesame target sequence. In some embodiments, the 3′ end of the targetsequence is directly adjacent to an AAA, GAA, CAA, or TAA sequence. Insome embodiments, the Cas9 protein comprises a combination of mutationsthat exhibit activity on a target sequence comprising a 5′-NAC-3′ PAMsequence at its 3′-end. In some embodiments, the combination ofmutations are present in any one of the clones listed in Table 2. Insome embodiments, the combination of mutations are conservativemutations of the clones listed in Table 2. In some embodiments, the Cas9protein comprises the combination of mutations of any one of the Cas9clones listed in Table 2.

TABLE 2 NAC PAM Clones MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO:18) T472I, R753G, K890E, D1332N, R1335Q, T1337N I1057S, D1135N, P1301S,R1335Q, T1337N T472I, R753G, D1332N, R1335Q, T1337N D1135N, E1219V,D1332N, R1335Q, T1337N T472I, R753G, K890E, D1332N, R1335Q, T1337NI1057S, D1135N, P1301S, R1335Q, T1337N T472I, R753G, D1332N, R1335Q,T1337N T472I, R753G, Q771H, D1332N, R1335Q, T1337N E627K, T638P, K652T,R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337NE627K, T638P, K652T, R753G, N803S, K959N, R1114G, D1135N, K1156E,E1219V, D1332N, R1335Q, T1337N E627K, T638P, V647I, R753G, N803S, K959N,G1030R, I1055E, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N E627K,E630G, T638P, V647A, G687R, N767D, N803S, K959N, R1114G, D1135N, E1219V,D1332G, R1335Q, T1337N E627K, T638P, R753G, N803S, K959N, R1114G,D1135N, E1219V, N1266H, D1332N, R1335Q, T1337N E627K, T638P, R753G,N803S, K959N, I1057T, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337NE627K, T638P, R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N,R1335Q, T1337N E627K, M631I, T638P, R753G, N803S, K959N, Y1036H, R1114G,D1135N, E1219V, D1251G, D1332G, R1335Q, T1337N E627K, T638P, R753G,N803S, V875I, K959N, Y1016C, R1114G, D1135N, E1219V, D1251G, D1332G,R1335Q, T1337N, I1348V K608R, E627K, T638P, V647I, R654L, R753G, N803S,T804A, K848N, V922A, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q,T1337N K608R, E627K, T638P, V647I, R753G, N803S, V922A, K959N, K1014N,V1015A, R1114G, D1135N, K1156N, E1219V, N1252D, D1332N, R1335Q, T1337NK608R, E627K, R629G, T638P, V647I, A711T, R753G, K775R, K789E, N803S,K959N, V1015A, Y1036H, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q,T1337N K608R, E627K, T638P, V647I, T740A, R753G, N803S, K948E, K959N,Y1016S, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N K608R,E627K, T638P, V647I, T740A, N803S, K948E, K959N, Y1016S, R1114G, D1135N,E1219V, N1286H, D1332N, R1335Q, T1337N I670S, K608R, E627K, E630G,T638P, V647I, R653K, R753G, I795L, K797N, N803S, K866R, K890N, K959N,Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N K608R, E627K,T638P, V647I, T740A, G752R, R753G, K797N, N803S, K948E, K959N, V1015A,Y1016S, R1114G, D1135N, E1219V, N1266H, D1332N, R1335Q, T1337N I570T,A589V, K608R, E627K, T638P, V647I, R654L, Q716R, R753G, N803S, K948E,K959N, Y1016S, R1114G, D1135N, E1207G, E1219V, N1234D, D1332N, R1335Q,T1337N K608R, E627K, R629G, T638P, V647I, R654L, Q740R, R753G, N803S,K959N, N990S, T995S, V1015A, Y1036D, R1114G, D1135N, E1207G, E1219V,N1234D, N1266H, D1332N, R1335Q, T1337N I562F, V565D, I570T, K608R,L625S, E627K, T638P, V647I, R654I, G752R, R753G, N803S, N808D, K959N,M1021L, R1114G, D1135N, N1177S, N1234D, D1332N, R1335Q, T1337N I562F,I570T, K608R, E627K, T638P, V647I, R753G, E790A, N803S, K959N, V1015A,Y1036H, R1114G, D1135N, D1180E, A1184T, E1219V, D1332N, R1335Q, T1337NI570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N,V1015A, R1114G, D1127A, D1135N, E1219V, D1332N, R1335Q, T1337N I570T,K608R, L625S, E627K, T638P, V647I, R654I, T703P, R753G, N803S, N808D,K959N, M1021L, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N I570S,K608R, E627K, E630G, T638P, V647I, R653K, R753G, I795L, N803S, K866R,K890N, K959N, Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337NI570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N,V1016A, R1114G, D1135N, E1219V, K1246E, D1332N, R1335Q, T1337N K608R,E627K, T638P, V647I, R654L, K673E, R753G, E790A, N803S, K948E, K959N,R1114G, D1127G, D1135N, D1180E, E1219V, N1286H, D1332N, R1335Q, T1337NK608R, L625S, E627K, T638P, V647I, R654I, I670T, R753G, N803S, N808D,K959N, M1021L, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337NE627K, M631V, T638P, V647I, K710E, R753G, N803S, N808D, K948E, M1021L,R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N, S1338T, H1349R

In some embodiments, the Cas9 protein comprises an amino acid sequencethat is at least 80% identical to the amino acid sequence of a Cas9protein as provided by any one of the variants of Table 2. In someembodiments, the Cas9 protein comprises an amino acid sequence that isat least 85%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence of a Cas9 protein as provided by any one of thevariants of Table 2.

In some embodiments, the Cas9 protein exhibits an increased activity ona target sequence that does not comprise the canonical PAM (5′-NGG-3′)at its 3′ end as compared to Streptococcus pyogenes Cas9 as provided bySEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits anactivity on a target sequence having a 3′ end that is not directlyadjacent to the canonical PAM sequence (5′-NGG-3′) that is at least5-fold increased as compared to the activity of Streptococcus pyogenesCas9 as provided by SEQ ID NO: 18 on the same target sequence. In someembodiments, the Cas9 protein exhibits an activity on a target sequencethat is not directly adjacent to the canonical PAM sequence (5′-NGG-3′)that is at least 10-fold, at least 50-fold, at least 100-fold, at least500-fold, at least 1,000-fold, at least 5,000-fold, at least10,000-fold, at least 50,000-fold, at least 100,000-fold, at least500,000-fold, or at least 1,000,000-fold increased as compared to theactivity of Streptococcus pyogenes as provided by SEQ ID NO: 18 on thesame target sequence. In some embodiments, the 3′ end of the targetsequence is directly adjacent to an AAC, GAC, CAC, or TAC sequence.

In some embodiments, the Cas9 protein comprises a combination ofmutations that exhibit activity on a target sequence comprising a5′-NAT-3′ PAM sequence at its 3′-end. In some embodiments, thecombination of mutations are present in any one of the clones listed inTable 3. In some embodiments, the combination of mutations areconservative mutations of the clones listed in Table 3. In someembodiments, the Cas9 protein comprises the combination of mutations ofany one of the Cas9 clones listed in Table 3.

TABLE 3 NAT PAM Clones MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO:18) K961E, H985Y, D1135N, K1191N, E1219V, Q1221H, A1320A, P1321S, R1335LD1135N, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L V743I,R753G, E790A, D1135N, G1218S, E1219V, Q1221H, A1227V, P1249S, N1286K,A1293T, P1321S, D1322G, R1335L, T1339I F575S, M631L, R654L, V748I,V743I, R753G, D853E, V922A, R1114G D1135N, G1218S, E1219V, Q1221H,A1227V, P1249S, N1286K, A1293T, P1321S, D1322G, R1335L, T1339I F575S,M631L, R654L, R664K, R753G, D853E, V922A, R1114G D1135N, D1180G, G1218S,E1219V, Q1221H, P1249S, N1286K, P1321S, D1322G, R1335L M631L, R654L,R753G, K797E, D853E, V922A, D1012A, R1114G D1135N, G1218S, E1219V,Q1221H, P1249S, N1317K, P1321S, D1322G, R1335L F575S, M631L, R654L,R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S,E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, M631L, R654L,R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S,E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, D596Y, M631L,R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G,G1218S, E1219V, Q1221H, P1249S, Q1256R, P1321S, D1322G, R1335L F575S,M631L, R654L, R664K, K710E, V750A, R753G, D853E, V922A, R1114G, Y1131C,D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335LF575S, M631L, K649R, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C,D1135N, K1156E, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G,R1335L F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C,D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335LF575S, M631L, R654L, R664K, R753G, D853E, V922A, I1057G, R1114G, Y1131C,D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, N1308D, P1321S, D1322G,R1335L M631L, R654L, R753G, D853E, V922A, R1114G, Y1131C, D1135N,E1150V, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335LM631L, R654L, R664K, R753G, D853E, I1057V, Y1131C, D1135N, D1180G,G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L M631L, R654L,R664K, R753G, I1057V, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V,Q1221H, P1249S, P1321S, D1332G, R1335L

Any of the above Cas9 variants displaying differential PAM specificitiesas compared to the canonical SpCas9 may be used in the herein disclosedprime editors.

P. Use of Prime Editing for Inserting Recombinase Target Sites

In another aspect, prime editing may be used to insert recombinase sites(or “recombinase recognition sequences”) into a desired genomic site.Insertion of recombinase sites provides a programmed location foreffecting site-specific genetic changes in a genome. Such geneticchanges can include, for example, genomic integration of a plasmid,genomic deletion or insertion, chromosomal translocations, and cassetteexchanges, among other genetic changes. These exemplary types of geneticchanges are illustrated in FIGS. 64B-64F. The installed recombinaserecognition sequences may then be used to conduct site-specificrecombination at that site to effectuate a variety of recombinationoutcomes, such as, excision, integration, inversion, or exchange of DNAfragments. For example, FIG. 65 illustrates the installation of arecombinase site that can then be used to integrate a DNA donor templatecomprising a GFP expression marker. Cells containing the integrated GFPexpression system into the recombinase site will fluoresce.

The mechanism of installing a recombinase site into the genome isanalogous to installing other sequences, such as peptide/protein and RNAtags, into the genome. A schematic exemplifying the installation of arecombinase target sequence is shows in FIG. 64(a). The process beginswith selecting a desired target locus into which the recombinase targetsequence will be introduced. Next, a prime editor fusion is provided(“RT-Cas9:gRNA”). Here, the “gRNA” refers to a PEgRNA, which can bedesigned using the principles described herein. The PEgRNA in variousembodiments will comprise an architecture corresponding to FIG. 3D(5′-[˜20-nt spacer]-[gRNA core]-[extension arm]-3′, wherein theextension arm comprises in the 3′ to 5′ direction, a primer binding site(“A”), an edit template (“B”), and a homology arm (“C”). The edittemplate (“B”) will comprise a sequence corresponding to a recombinasesite, i.e., a single strand RNA of the PEgRNA that codes for acomplementary single strand DNA that is either the sense or theantisense strand of the recombinase site and which is incorporated intothe genomic DNA target locus through the prime editing process.

In various aspects, the present disclosure provides for the use of a PEto introduce recombinase recognition sequences at high-value loci inhuman or other genomes, which, after exposure to site-specificrecombinase(s), will direct precise and efficient genomic modifications(FIG. 64 ). In various embodiments show in FIG. 64 , PE may be used to(b) insert a single SSR target for use as a site for genomic integrationof a DNA donor template. (c) shows how a tandem insertion of SSR targetsites can be used to delete a portion of the genome. (d) shows how atandem insertion of SSR target sites can be used to invert a portion ofthe genome. (e) shows how the insertion of two SSR target sites at twodistal chromosomal regions can result in chromosomal translocation. (f)shows how the insertion of two different SSR target sites in the genomecan be used to exchange a cassette from a DNA donor template. Each ofthe types of genome modifications are envisioned by using PE to insertSSR Tagetes, but this list also is not meant to be limiting.

PE-mediated introduction of recombinase recognition sequences could beparticularly useful for the treatment of genetic diseases which arecaused by large-scale genomic defects, such as gene loss, inversion, orduplication, or chromosomal translocation¹⁻⁷ (Table 6). For example,Williams-Beuren syndrome is a developmental disorder caused by adeletion of 24 in chromosome 721. No technology exists currently for theefficient and targeted insertion of multiple entire genes in livingcells (the potential of PE to do such a full-length gene insertion iscurrently being explored but has not yet been established); however,recombinase-mediated integration at a target inserted by PE offers oneapproach towards a permanent cure for this and other diseases. Inaddition, targeted introduction of recombinase recognition sequencescould be highly enabling for applications including generation oftransgenic plants, animal research models, bioproduction cell lines, orother custom eukaryotic cell lines. For example, recombinase-mediatedgenomic rearrangement in transgenic plants at PE-specific targets couldovercome one of the bottlenecks to generating agricultural crops withimproved properties^(8,9).

TABLE 6 Examples of genetic diseases linked to large-scale genomicmodifications that could be repaired through PE-based installation ofrecombinase recognition sequences. DISEASE CAUSE TRISOMY 17P GENEDUPLICATION CHARCOT-MARIE- GENE DUPLICATION TOOTH DISEASE TYPE ISMITH-MAGENIS GENE DELETION SYNDROME WILLIAMS-BEUREN GENE DELETIONSYNDROME DE LA CHAPELLE CHROMOSOMAL SYNDROME TRANSLOCATION DOWN SYNDROMECHROMOSOMAL (SOME FORMS) TRANSLOCATION HEMOPHILIA A GENE INVERSIONHUNTER SYNDROME GENE INVERSION

A number of SSR family members have been characterized and theirrecombinase recognition sequences described, including natural andengineered tyrosine recombinases (Table 7), large serine integrases(Table 8), serine resolvases (Table 9), and tyrosine integrases (Table10). Modified target sequences that demonstrate enhanced rates ofgenomic integration have also been described for several SSRs²²⁻³⁰. Inaddition to natural recombinases, programmable recombinases withdistinct specificities have been developed³¹⁻⁴⁰. Using PE, one or moreof these recognition sequences could be introduced into the genomic at aspecified location, such as a safe harbor locus⁴¹⁻⁴³, depending on thedesired application.

For example, introduction of a single recombinase recognition sequencein the genome by prime editing would result in integrative recombinationwith a DNA donor template (FIG. 64 b ). Serine integrases, which operaterobustly in human cells, may be especially well-suited for geneintegration^(44,45).

Additionally, introduction of two recombinase recognition sequencescould result in deletion of the intervening sequence, inversion of theintervening sequence, chromosomal translocation, or cassette exchange,depending on the identity and orientation of the targets (FIGS.64C-64F). By choosing endogenous sequences that already closely resemblerecombinase targets, the scope of editing required to introduce thecomplete recombinase target would be reduced.

Finally, several recombinases have been demonstrated to integrate intohuman or eukaryotic genomes at natively occurring pseudosites⁴⁶⁻⁶⁴. PEediting could be used to modify these loci to enhance rates ofintegration at these natural pseudosites, or alternatively, to eliminatepseudosites that may serve as unwanted off-target sequences.

This disclosure describes a general methodology for introducingrecombinase target sequences in eukaryotic genomes using PE, theapplications of which are nearly limitless. The genome editing reactionsare intended for use with “prime editor,” a chimeric fusion of aCRISPR/Cas9 protein and a reverse-transcriptase domain, which utilizes acustom prime editing guide RNA (PEgRNA). By extension, Cas9 tools andhomology-directed repair (HDR) pathways may also be exploited tointroduce recombinase recognition sequences through DNA templates bylowering the rates of indels using several techniques⁶⁵⁻⁶⁷. Aproof-of-concept experiment in human cell culture is shown in FIG. 65 .

The following several tables are cited in the above description relatingto PE-directed installation of recombinase recognition sequences andprovide a listing of exemplary recombinases that may be used, and theircognate recombinase recognition sequences that may be installed by PE.

TABLE 7 Tyrosine recombinases and SSR target sequences. RecombinaseRecombinase recognition sequence Name CreATAACTTCGTATAGCATACATTATACGAAGTTAT loxP (SEQ ID NO: 517) DreTAACTTTAAATAATGCCAATTATTTAAAGTTA (SEQ rox ID NO: 518) VCreTCAATTTCTGAGAACTGTCATTCTCGGAAATTGA loxV (SEQ ID NO: 519) SCreCTCGTGTCCGATAACTGTAATTATCGGACATGAT loxS (SEQ ID NO: 520) FlpGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC FRT (SEQ ID NO: 521) B2GAGTTTCATTAAGGAATAACTAATTCCCTAATGAA loxB ACTC (SEQ ID NO: 522) B3GGTTGCTTAAGAATAAGTAATTCTTAAGCAACC loxB3 (SEQ ID NO: 523) KwACGAAAAATGGTAAGGAATAGACCATTCCTTACC ATTTTTGGT (SEQ ID NO: 524) RTTGATGAAAGAATAACGTATTCTTTCATCAA (SEQ RS ID NO: 525) TD1-40GTGCGTCAAATAATAACGTATTATTTGACACTT TDRS (SEQ ID NO: 526) VikaAATAGGTCTGAGAACGCCCATTCTCAGACGTATT vox (SEQ ID NO: 527) NigriTGAATGTCCTATAATTACACTTATAGGACATTCA nox (SEQ ID NO: 528) PantoGAAACTTTAAATAATAAGTCTTATTTAAAGTTTC pox (SEQ ID NO: 529) KdAAACGATATCAGACATTTGTCTGATAATGCTTCA loxKTTATCAGACAAATGTCTGATATCGTTT (SEQ ID NO: 530) FreATATATACGTATATAGACATATATACGTATATAT loxH (SEQ ID NO: 531) CreALSHGATAACTCTATATAATGTATGCTATATAGAGTTAT loxM7 (SEQ ID NO: 532) TreACAACATCCTATTACACCCTATATGCCAACATGG loxLTR (SEQ ID NO: 533) Brec1AACCCACTGCTTAAGCCTCAATAAAGCTTGCCTT loxBTR (SEQ ID NO: 534) Cre-R3M3GATACAACGTATATACCTTTCTATACGTTGTTTA loxK2 (SEQ ID NO: 535)

TABLE 8 Large serine integrases and SSR target sequences.Recombinase recognition sequence Recombinase recognition sequenceRecombinase Left Right Bxb1 GGTTTGTCTGGTCAACCACCGCGGCTTGTCGACGACGGCGGTCTCC GGTCTCAGTGGTGTACGGTACAGTCGTCAGGATCAT (SEQ ID NO: AACC (SEQ ID NO: 536) 537) phiC31GTGCCCCAACTGGGGTAACCTT TGCGGGTGCCAGGGCGTGCCCTTG TGAGTTCTCTCAGTTGGGGGGGCTCCCCGGGCGCGTACTCC (SEQ (SEQ ID NO: 538) ID NO: 539) R4TGTTCCCCAAAGCGATACCACT GCATGTTCCCCAAAGCGATACCAC TGAAGCAGTGGTACTGCTTGTGTTGAAGCAGTGGTACTGCTTGTGG GGTACA (SEQ ID NO: 540)GTACACTCTGCGGGTG (SEQ ID NO: 541) phiBT1 GGTGCTGGGTTGTTGTCTCTGGCAGGTTTTTGACGAAAGTGATCCA ACAGTGATCCATGGGAAACTAGATGATCCAG (SEQ ID NO: 543) CTCAGCACC (SEQ ID NO: 542) MJ1 (phiFC1)ATTTTAGGTATATGATTTTGTT CAAAGGATCACTGAATCAAAAGTA TATTAGTGTAAATAACACTATGTTGCTCATCCACGCGAAA (SEQ ID TACCTAAAAT (SEQ ID NO: 544) NO: 545) MR11TTTGTGCGGAACTACGAACAGT CGAAAATGTATGGAGGCACTTGTA TCATTAATACGAAGTGTACAATCAATATAGGATGTATACCTTCGA ACTTCCATACAA (SEQ ID NO:AGACACTT (SEQ ID NO: 547) 546) TP901-1 GAGTTTTTATTTCGTTTATTTCAATGCCAACACAATTAACATCTCAA ATTAAGGTAACTAAAAAACTC TCAAGGTAAATGCTTTTTGCTTTTTCTTTTAAGG (SEQ ID NO: 548) TTGC (SEQ ID NO: 549) A118TTCCTCGTTTTCTCTCGTTGGA TTTCGGATCAAGCTATGAAGGACG AGAAGAAGAAACGAGAAACAAAGAGGGAACTAAA (SEQ ID NO: (SEQ ID NO: 550) 551) U153TTCCTCGTTTTCTCTCGTTGGAC TTTCGGATCAAGCTATGAAGGACG GGAAACGAATCGAGAAA (SEQCAAAGAGGGAACTAAA (SEQ ID ID NO: 552) NO: 553) phiRV1GTAGTGTATCTCACAGGTCCAC GAAGGTGTTGGTGCGGGGTTGGCC GGTTGGCCGTGGACTGCTGAAGTGGTCGAGGTGGGGT (SEQ ID NO: GAACATTCC (SEQ ID NO: 554) 555) phi370.1AAAAAAATACAGCGTTTTTCAT TTGTAAAGGAGACTGATAATGGCA GTACAACTATACTAGTTGTAGTTGTACAACTATACTCGTCGGTAAA GCCTAAAA (SEQ ID NO: 556)AAGGCA (SEQ ID NO: 557) TG1 TCCAGCCCAACAGTGTTAGTCTGATCAGCTCCGCGGGCAAGACCTT TTGCTCTTACCCAGTTGGGCGG TCTCCTTCACGGGGTGGAAGGTCGA (SEQ ID NO: 558) (SEQ ID NO: 559) WB CTAGTTTTAAAGTTGGTTATTACGGAAGGTAGCGTCAACGATAGGT GTTACTGTGATATTTATCACGGGTAACTGTCGTGTTTGTAACGGTAC TACCCAATAACCAATGAAT TTCCAACAGCTGGCGCCGCCAC(SEQ ID NO: 560) (SEQ ID NO: 561) BL3 CAATGAAAAACTAGGCATGTATTTCCACAGACAACTCACGTGGAG GAAGTTGTTTGT (SEQ ID NO:GTAGTCAC (SEQ ID NO: 563) 562) SprA TGTAGTAAGTATCTTAATATACCACCCATTGTGTTCACAGGAGATA AGCTTTATCTGTTTTTTAAGATCAGCTTTATCTGTACTGATATTAAT ACTTACTACTTT (SEQ ID NO:GACATGCTG (SEQ ID NO: 565) 564) phiJoe AGTTGTGGCCATGTGTCCATCTATCTGGATGTGGGTGTCCATCTGCG GGGGGCAGATGGAGACGGGGT GGCAGACGCCGCAGTCGAAGCACGCACA (SEQ ID NO: 566) G (SEQ ID NO: 567) — — ACCTTGATCTCGGTGTCCATCGCCGGGCAGACGCCGCAGTCGAAGCACG G (SEQ ID NO: 568) phiK38 CCCTAATACGCAAGTCGATAAGAGCGCCGGATCAGGGAGTGGACG CTCTCCTGGGAGCGTTGACAAC GCCTGGGAGCGCTACACGCTGTGGTTGCGCACCCTGATCTG (SEQ CTGCGGTCGGTGC (SEQ ID NO: 570) ID NO: 569) Int2GCTCATGTATGTGTCTACGCGA GGACGGCGCAGAAGGGGAGTAGCT GATTCTCGCCCGAGAACTTCTGCTTCGCCGGACCGTCGACATACTG CAAGGCACTGCTCTTGGCTCTCAGCTCGTC (SEQ ID NO: 572) (SEQ ID NO: 571) Int3 ATGGATAAAAAAATACAGCGTGTTTGTAAAGGAGACTGATAATGG TTTTCATGTACAACTATACTAG CATGTACAACTATACTCGTCGGTATTGTAGTGCCTAAATAATGCTT AAAAGGCATCTTAT (SEQ ID NO: (SEQ ID NO: 573) 574)Int4 AAAAATTACAAAGTTTTCAACC TTCCAAAGAGCGCCCAACGCGACCCTTGATTTGAATTAGCGGTCAA TGAAATTTGAATAAGACTGCTGCTT ATAATTTGTAATTCGTTT (SEQGTGTAAAGGCGATGATT (SEQ ID ID NO: 575) NO: 576) Int7GTGTTATAAACCTGTGTGAGAG AGACGAGAAACGTTCCGTCCGTCT TTAAGTTTACATGCCTAACCTTGGGTCAGTTGGGCAAAGTTGATGA AACTTTTACGCAGGTTCAGCTTCCGGGTCGTCCGTT (SEQ ID NO: (SEQ ID NO: 577) 578) Int8TTAATAAACTATGGAAGTATGT CAATCATCAGATAACTATGGCGGC ACAGTCTTGCAATGTTGAGTGAACGTGCATTAACCACGGTTGTATCC ACAAACTTCCATAATAAAATCGTCTAAAGTACTCGT (SEQ ID NO: (SEQ ID NO: 579) 580) Int9GTGGTTGTTTTTGTTGGAAGTG TTTATATTGCGAAAAATAATTGGC TGTATCAGGTATCTGCATAGTTGAACGAGGTAACTGGATACCTCAT ATTCCGAACTTCCAATTA (SEQCCGCCAATTAAAATTTG (SEQ ID ID NO: 581) NO: 582) Int10GGAAAATATAAATAATTTTAGT AGCACGCTGATAATCAGCAAGACC AACCTACATCTCAATCAAGGATACCAACATTTCCACCAATGTAAAA AGTAAAACTCTCACTCTT (SEQGCTTTAACCTTAGC (SEQ ID NO: ID NO: 583) 584) Int11 GTTTATATGTTTACTAATAAGAATGGATTTTGCAGATTCCCAGATGC CGCTCTCAACCCATAAAGTCTTCCCTACAGAAAGAGGTACAAAACA ATTAGTAAACATATTTCAACTTTTATTGGAATTAATT (SEQ ID NO: (SEQ ID NO: 585) 586) Int12TTTTTGTATGTTAGTTGTGTCA GTTCGTGGTAACTATGGGTGGTAC CTGGGTAGACCTAAATAGTGAAGGTGCCACATTAGTTGTACCATTT CACAACTGCTATTAAAATTTAAATGTTTATGTGGTTAAC (SEQ ID (SEQ ID NO: 587) NO: 588) Int13CAATAACGGTTGTATTTGTAGA GCATACATTGTTGTTGTTTTTCCAG ACTTGACCAGTTGTTTTAGTAAATCCAGTTGGTCCTGTAAATATAA CATAAATACAACTCCGAATAGCAATCCATGTGAGT (SEQ ID NO: (SEQ ID NO: 589) 590) LIGTTTAGTATCTCGTTATCTCTC TAACTTTTTCGGATCGAGTTATGAT GTTGGAGGGAGAAGAAACGGGGGACGTAAAGAGGGAACAAAGCA ATACCAAAA (SEQ ID NO: 591) TCTA (SEQ ID NO: 592)Peaches TAGTTTCCAATGTTACAGGAAC CGGTCTCCATCGGGATCTGCTGATCTGCTGGCAGAATCCAACACATT GAGCAGCATGCCGACCA (SEQ IDGGAAGTCG (SEQ ID NO: 593) NO: 594) Bxz2 TAACCGCAAGTGTACATCCCTCCGGTCTCCATCGGGATCTGCTGATC GGCTGGCCGAGACAAGTACAGGAGCAGCATGCCGACCA (SEQ ID TTGCGACAG (SEQ ID NO: 595) NO: 596) SV1ATGTGGTCCTTTAGATCCACTG CATCAGGGCGGTCAGGCCGTAGAT ACGTGGGTCAGTGTCTCTAAAGGTGGAAGAAACGGCAGCACGGCG GACTCGCG (SEQ ID NO: 597)AGGACG (SEQ ID NO: 598)

TABLE 9 Serine resolvases and SSR target sequences.Recombinase recognition sequence Recombinase recognition sequenceResolvase Left Right Gin CGTTTCCTGTAAACCGAGGTTTTGCGTTTCCTGTAAACCGAGGTTTTG GATAAACA (SEQ ID NO: 599)GATAATGG (SEQ ID NO: 600) Cin GAGTTCTCTTAAACCAAGGTTTAGAGTTCTCTTAAACCAAGGTATT GGATTGAAA (SEQ ID NO: 601)GGATAACAG (SEQ ID NO: 602) Hin TGGTTCTTGAAAACCAAGGTTTTTAAATTTTCCTTTTTGGAAGGTTTT GATAAAGC (SEQ ID NO: 603)TGATAACCA (SEQ ID NO: 604) Min GCCTTCCCCTAAACCAACGTTTTTGCCTTCCCCCAAACCAAGGTAAT ATGCCGCC (SEQ ID NO: 605)CAAGAACGC (SEQ ID NO: 606) Sin TTGTGAAATTTGGGTACACCCTACGTATGATTAGGGTGTATATTAA ATCATACAA (SEQ ID NO: 607) TTT (SEQ ID NO: 608)

TABLE 10 Tyrosine integrases and target sequences. Integrase attP attBHK022 CAAATGATTTTATTTTGACTAATAA GCACTTTAGGTGAAAAAGGTTGACCTACTTACATTAATTTACTGAT T (SEQ ID NO: 610) AATTAAAGAGATTTTAAATATACAACTTATTCACCTAAAGGATGACAAAA (SEQ ID NO: 609) TAACATTAATCACTTAAAAATCATCGCATTACACTAATCTGTGGTTAAAT GATAGACTACATAATGCGACAAAACGCAACATATCCAGTCACTATGAAT CAACTACTTAGATAGTATTAGTGAC CT (SEQ ID NO: 611)P22 CTAAGTGGTTTGGGACAAAAATGGG GCAGCGCATTCGTAATGCGAACATACAAATCTTTGCATCGGTTTG AGGTCGT (SEQ ID NO: 613)CAAGGCTTTGCATGTCTTTCGAAGA TGGGACGTGTGAGCGCAGGTATGACGTGGTATGTGTTGACTTAAAAGGTA GTTCTTATAATTCGTAATGCGAAGGTCGTAGGTTCGACTCCTATTATCGG CACCAGTTAAATCAAATACTTACGTATTATTCGTGCCTTCCTTATTTTTAC TGTGGGACATATTTGGGACAGAAGTACCAAAAA (SEQ ID NO: 612) L5 GCGATCCCCATCCGCGACGTGCCAAGAGCGGGCGACGGGAATCG CTAGGTCTCCTCTCGTCGTGAACAA AACCCGCGTAGCTAGTTTGGGGCTACCGGGTTGCAACTCCTGTGC AAGA (SEQ ID NO: 615)AACTCTCAGGCTTCAACGCGCTTCT ACGACCTGCAATTTCTTTCCACTTAGAGGATGCAGCCGAGAGGGGTAAA AACCTATCTTGACCGGCCCATATGTGGTCGGCAGACACCCATTCTTCCAA ACTAGCTACGCGGGTTCGATTCCCGTCGCCCGCTCCGCTGGTCAGAGGGT GTTTTCGCCCTCTGGCCATTTTTCTTTCCAGGGGTCTGCAACTCTTGTGCG ACTCTTCTGACCTGGGCATACGCGGTTGCAACGCATCCCTGATCTGGCTA CTTTCGATGCTGACAAACGAATAGAGCCCCCCGCCTGCGCGAACAGACG AGGGGCATTCACA (SEQ ID NO: 614)

In various other aspects, the present disclosure relates to methods ofusing PE to install one or more recombinase recognition sequence andtheir use in site-specific recombination.

In some embodiments, the site-specific recombination may effectuate avariety of recombination outcomes, such as, excision, integration,inversion, or exchange of DNA fragments.

In some embodiments, the methods are useful for inducing recombinationof or between two or more regions of two or more nucleic acid (e.g.,DNA) molecules. In other embodiments, the methods are useful forinducing recombination of or between two or more regions in a singlenucleic acid molecule (e.g., DNA).

In some embodiments, the disclosure provides a method for integrating adonor DNA template by site-specific recombination, comprising: (a)installing a recombinase recognition sequence at a genomic locus byprime editing; (b) contacting the genomic locus with a DNA donortemplate that also comprises the recombinase recognition sequence in thepresence of a recombinase.

In other embodiments, the disclosure provides a method for deleting agenomic region by site-specific recombination, comprising: (a)installing a pair of recombinase recognition sequences at a genomiclocus by prime editing; (b) contacting the genomic locus with arecombinase, thereby catalyzing the deletion of the genomic regionbetween the pair of recombinase recognition sequences.

In yet other embodiments, the disclosure provides a method for invertinga genomic region by site-specific recombination, comprising: (a)installing a pair of recombinase recognition sequences at a genomiclocus by prime editing; (b) contacting the genomic locus with arecombinase, thereby catalyzing the inversion of the genomic regionbetween the pair of recombinase recognition sequences.

In still other embodiments, the disclosure provides a method forinducing chromosomal translocation between a first genomic site and asecond genomic site, comprising: (a) installing a first recombinaserecognition sequence at a first genomic locus by prime editing; (b)installing a second recombinase recognition sequence at a second genomiclocus by prime editing; (c) contacting the first and the second genomicloci with a recombinase, thereby catalyzing the chromosomaltranslocation of the first and second genomic loci.

In other embodiments, the disclosure provides a method for inducingcassette exchange between a genomic locus and a donor DNA comprising acassette, comprising: (a) installing a first recombinase recognitionsequence at a first genomic locus by prime editing; (b) installing asecond recombinase recognition sequence at a second genomic locus byprime editing; (c) contacting the first and the second genomic loci witha donor DNA comprising a cassette that is flanked by the first andsecond recombinase recognition sequences and a recombinase, therebycatalyzing the exchange of the flanked genomic locus and the cassette inthe DNA donor.

In various embodiments involving the insertion of more than onerecombinase recognition sequences in the genome, the recombinaserecognition sequences can be the same or different. In some embodiments,the recombinase recognition sequences are the same. In otherembodiments, that recombinase recognition sequences are different.

In various embodiments, the recombinase can be a tyrosine recombinase,such as Cre, Dre, Vcre, Scre, Flp, B2, B3, Kw, R, TD1-40, Vika, Nigri,Panto, Kd, Fre, Cre(ALSHG), Tre, Brec1, or Cre-R3M3, as shown in Table7. In such embodiments, the recombinase recognition sequence may be anRRS of Table 7 that corresponds to the recombinase under use.

In various other embodiments, the recombinase can be a large serinerecombinase, such as Bxb1, PhiC31, R4, phiBT1, MJ1, MR11, TP901-1, A118,V153, phiRV1, phi370.1, TG1, WB, BL3, SprA, phiJoe, phiK38, Int2, Int3,Int4, Int7, Int8, Int9, Int10, Intl 1, Int12, Int13, L1, peaches, Bxz2,or SV1, as shown in Table 8. In such embodiments, the recombinaserecognition sequence may be an RRS of Table 8 that corresponds to therecombinase under use.

In still other embodiments, the recombinase can be a serine recombinase,such as Bxb1, PhiC31, R4, phiBT1, MJ1, MR11, TP901-1, A118, V153,phiRV1, phi370.1, TG1, WB, BL3, SprA, phiJoe, phiK38, Int2, Int3, Int4,Int7, Int8, Int9, Int10, Intl 1, Int12, Int13, L1, peaches, Bxz2, orSV1, as shown in Table 8. In such embodiments, the recombinaserecognition sequence may be an RRS of Table 8 that corresponds to therecombinase under use.

In other embodiments, the recombinase can be a serine resolvase, such asGin, Cin, Hin, Min, or Sin, as shown in Table 9. In such embodiments,the recombinase recognition sequence may be an RRS of Table 9 thatcorresponds to the recombinase under use.

In various other embodiments, the recombinase can be a tyrosineintegrase, such as HK022, P22, or L5, as shown in Table 10. In suchembodiments, the recombinase recognition sequence may be an RRS of Table10 that corresponds to the recombinase under use.

In some embodiments, any of the methods for site-specific recombinationwith PE can be performed in vivo or in vitro. In some embodiments, anyof the methods for site-specific recombination are performed in a cell(e.g., recombine genomic DNA in a cell). The cell can be prokaryotic oreukaryotic. The cell, such as a eukaryotic cell, can be in anindividual, such as a subject, as described herein (e.g., a humansubject). The methods described herein are useful for the geneticmodification of cells in vitro and in vivo, for example, in the contextof the generation of transgenic cells, cell lines, or animals, or in thealteration of genomic sequence, e.g., the correction of a geneticdefect, in a cell in a subject.

REFERENCES CITED FOR SECTION L

Each of the following references are cited in Example 17, each of whichare incorporated herein by reference.

-   1. Feuk, L. Inversion variants in the human genome: role in disease    and genome architecture. Genome Med 2, 11 (2010).-   2. Zhang, F., Gu, W., Hurles, M. E. & Lupski, J. R. Copy number    variation in human health, disease, and evolution. Annu Rev Genomics    Hum Genet 10, 451-481 (2009).-   3. Shaw, C. J. & Lupski, J. R. Implications of human genome    architecture for rearrangement-based disorders: the genomic basis of    disease. Hum Mol Genet 13 Spec No 1, R57-64 (2004).-   4. Carvalho, C. M., Zhang, F. & Lupski, J. R. Evolution in health    and medicine Sackler colloquium: Genomic disorders: a window into    human gene and genome evolution. Proc Natl Acad Sci USA 107 Suppl 1,    1765-1771 (2010).-   5. Rowley, J. D. Chromosome translocations: dangerous liaisons    revisited. Nat Rev Cancer 1, 245-250 (2001).-   6. Aplan, P. D. Causes of oncogenic chromosomal translocation.    Trends Genet 22, 46-55 (2006).-   7. McCarroll, S. A. & Altshuler, D. M. Copy-number variation and    association studies of human disease. Nat Genet 39, S37-42 (2007).

[8] Methods of Treatment

The instant disclosure provides methods for the treatment of a subjectdiagnosed with a disease associated with or caused by a point mutation,or other mutations (e.g., deletion, insertion, inversion, duplication,etc.) that can be corrected by the prime editing system provided herein,as exemplified, but not limited to prion disease (e.g., Example 5herein), trinucleotide repeat expansion disease (e.g., Example 3herein), or CDKL5 Deficiency Disorder (CDD) (e.g., Example 23 herein).

Virtually any disease-causing genetic defect may be repaired by usingprime editing, which includes the selection of an appropriate primeeditor fusion protein (including a napDNAbp and a polymerase (e.g., areverse transcriptase), and designing of an appropriate PEgRNA designedto (a) target the appropriate target DNA containing an edit site, and(b) provide a template for the synthesis of a single strand of DNA fromthe 3′ end of the nick site that includes the desired edit whichdisplaces and replaces the endogenous strand immediately downstream ofthe nick site. Prime editing can be used, without limitation, to (a)install mutation-correcting changes to a nucleotide sequence, (b)install protein and RNA tags, (c) install immunoepitopes on proteins ofinterest, (d) install inducible dimerization domains in proteins, (e)install or remove sequences to alter that activity of a biomolecule, (f)install recombinase target sites to direct specific genetic changes, and(g) mutagenesis of a target sequence by using an error-prone RT.

The method of treating a disorder can involve as an early step thedesign of an appropriate PEgRNA and prime editor fusion protein inaccordance with the methods described herein, which include a number ofconsiderations that may be taken into account, such as:

-   -   (a) the target sequence, i.e., the nucleotide sequence in which        one or more nucleobase modifications are desired to be installed        by the prime editor;    -   (b) the location of the cut site within the target sequence,        i.e., the specific nucleobase position at which the prime editor        will induce a single-stand nick to create a 3′ end RT primer        sequence on one side of the nick and the 5′ end endogenous flap        on the other side of the nick (which ultimately is removed by        FEN1 or equivalent thereto and replaced by the 3′ ssDNA flap.        The cut site creates the 3′ end primer sequence which becomes        extended by the polymerase of the PE fusion protein (e.g., a RT        enzyme) during RNA-dependent DNA polymerization to create the 3′        ssDNA flap containing the desired edit, which then replaces the        5′ endogenous DNA flap in the target sequence.    -   (c) the available PAM sequences (including the canonical SpCas9        PAM sites, as well as non-canonical PAM sites recognized by Cas9        variants and equivalents with expanded or differing PAM        specificities);    -   (d) the spacing between the available PAM sequences and the        location of the cut site in the PAM strand;    -   (e) the particular Cas9, Cas9 variant, or Cas9 equivalent of the        prime editor available to be used (which in part is dictated by        the available PAM);    -   (f) the sequence and length of the primer binding site;    -   (g) the sequence and length of the edit template;    -   (h) the sequence and length of the homology arm;    -   (i) the spacer sequence and length; and    -   (j) the gRNA core sequence.

A suitable PEgRNA, and optionally a nicking-sgRNA design guide forsecond-site nicking, can be designed by way of the following exemplarystep-by-step set of instructions which takes into account one or more ofthe above considerations. The steps reference the examples shown inFIGS. 70A-70I.

-   -   1. Define the target sequence and the edit. Retrieve the        sequence of the target DNA region (˜200 bp) centered around the        location of the desired edit (point mutation, insertion,        deletion, or combination thereof). See FIG. 70A.    -   2. Locate target PAMs. Identify PAMs in the proximity to the        desired edit location. PAMs can be identified on either strand        of DNA proximal to the desired edit location. While PAMs close        to the edit position are preferred (i.e., wherein the nick site        is less than 30 nt from the edit position, or less than 29 nt,        28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt,        19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt,        10 nt, 9 nt, 8 nt, 7 nt, 6 nt, 5 nt, 4 nt, 3 nt, or 2 nt from        the edit position to the nick site), it is possible to install        edits using protospacers and PAMs that place the nick ≥30 nt        from the edit position. See FIG. 70B.    -   3. Locate the nick sites. For each PAM being considered,        identify the corresponding nick site and on which strand. For Sp        Cas9 H840A nickase, cleavage occurs in the PAM-containing strand        between the 3^(th) and 4^(th) bases 5′ to the NGG PAM. All        edited nucleotides must exist 3′ of the nick site, so        appropriate PAMs must place the nick 5′ to the target edit on        the PAM-containing strand. In the example shown below, there are        two possible PAMs. For simplicity, the remaining steps will        demonstrate the design of a PEgRNA using PAM 1 only. See FIG.        70C.    -   4. Design the spacer sequence. The protospacer of SpCas9        corresponds to the 20 nucleotides 5′ to the NGG PAM on the        PAM-containing strand. Efficient Pol III transcription        initiation requires a G to be the first transcribed nucleotide.        If the first nucleotide of the protospacer is a G, the spacer        sequence for the PEgRNA is simply the protospacer sequence. If        the first nucleotide of the protospacer is not a G, the spacer        sequence of the PEgRNA is G followed by the protospacer        sequence. See FIG. 70D.    -   5. Design a primer binding site (PBS). Using the starting allele        sequence, identify the DNA primer on the PAM-containing strand.        The 3′ end of the DNA primer is the nucleotide just upstream of        the nick site (i.e. the 4^(th) base 5′ to the NGG PAM for Sp        Cas9). As a general design principle for use with PE2 and PE3, a        PEgRNA primer binding site (PBS) containing 12 to 13 nucleotides        of complementarity to the DNA primer can be used for sequences        that contain ˜40-60% GC content. For sequences with low GC        content, longer (14- to 15-nt) PBSs should be tested. For        sequences with higher GC content, shorter (8- to 11-nt) PBSs        should be tested. Optimal PBS sequences should be determined        empirically, regardless of GC content. To design a length-p PBS        sequence, take the reverse complement of the first p nucleotides        5′ of the nick site in the PAM-containing strand using the        starting allele sequence. See FIG. 70E.    -   6. Design an RT template (or DNA synthesis template). The RT        template (or DNA synthesis template where the polymerase is not        reverse transcriptase) encodes the designed edit and homology to        the sequence adjacent to the edit. In one embodiment, these        regions correspond to the DNA synthesis template of FIG. 3D and        FIG. 3E, wherein the DNA synthesis template comprises the “edit        template” and the “homology arm.” Optimal RT template lengths        vary based on the target site. For short-range edits (positions        +1 to +6), it is recommended to test a short (9 to 12 nt), a        medium (13 to 16 nt), and a long (17 to 20 nt) RT template. For        long-range edits (positions +7 and beyond), it is recommended to        use RT templates that extend at least 5 nt (preferably 10 or        more nt) past the position of the edit to allow for sufficient        3′ DNA flap homology. For long-range edits, several RT templates        should be screened to identify functional designs. For larger        insertions and deletions (≥5 nt), incorporation of greater 3′        homology (˜20 nt or more) into the RT template is recommended.        Editing efficiency is typically impaired when the RT template        encodes the synthesis of a G as the last nucleotide in the        reverse transcribed DNA product (corresponding to a C in the RT        template of the PEgRNA). As many RT templates support efficient        prime editing, avoidance of G as the final synthesized        nucleotide is recommended when designing RT templates. To design        a length-r RT template sequence, use the desired allele sequence        and take the reverse complement of the first r nucleotides 3′ of        the nick site in the strand that originally contained the PAM.        Note that compared to SNP edits, insertion or deletion edits        using RT templates of the same length will not contain identical        homology. See FIG. 70F.    -   7. Assemble the full PEgRNA sequence. Concatenate the PEgRNA        components in the following order (5′ to 3′): spacer, scaffold,        RT template and PBS. See FIG. 70G.    -   8. Designing nicking-sgRNAs for PE3. Identify PAMs on the        non-edited strand upstream and downstream of the edit. Optimal        nicking positions are highly locus-dependent and should be        determined empirically. In general, nicks placed 40 to 90        nucleotides 5′ to the position across from the PEgRNA-induced        nick lead to higher editing yields and fewer indels. A nicking        sgRNA has a spacer sequence that matches the 20-nt protospacer        in the starting allele, with the addition of a 5′-G if the        protospacer does not begin with a G. See FIG. 70H.    -   9. Designing PE3b nicking-sgRNAs. If a PAM exists in the        complementary strand and its corresponding protospacer overlaps        with the sequence targeted for editing, this edit could be a        candidate for the PE3b system. In the PE3b system, the spacer        sequence of the nicking-sgRNA matches the sequence of the        desired edited allele, but not the starting allele. The PE3b        system operates efficiently when the edited nucleotide(s) falls        within the seed region (˜10 nt adjacent to the PAM) of the        nicking-sgRNA protospacer. This prevents nicking of the        complementary strand until after installation of the edited        strand, preventing competition between the PEgRNA and the sgRNA        for binding the target DNA. PE3b also avoids the generation of        simultaneous nicks on both strands, thus reducing indel        formation significantly while maintaining high editing        efficiency. PE3b sgRNAs should have a spacer sequence that        matches the 20-nt protospacer in the desired allele, with the        addition of a 5′ G if needed. See FIG. 70I.

The above step-by-step process for designing a suitable PEgRNA and asecond-site nicking sgRNA is not meant to be limiting in any way. Thedisclosure contemplates variations of the above-described step-by-stepprocess which would be derivable therefrom by a person of ordinary skillin the art.

Once a suitable PEgRNA and PE fusion protein are selected/designed, theymay be administered by a suitable methodology, such as by vector-basedtransfection (in which one or more vectors comprising DNA encoding thePEgRNA and the PE fusion protein and which are expressed within a cellupon transfection with the vectors), direct delivery of the PE fusionprotein complexed with the PEgRNA (e.g., RNP delivery) in a deliveryformat (e.g., lipid particles, nanoparticles), or by a mRNA-baseddelivery system. Such methods are described herein in the presentdisclosure and any know method may be utilized.

The PEgRNA and PE fusion protein (or together, referred to as the PEcomplex) can be delivered to a cell in a therapeutically effectiveamount such that upon contacting the target DNA of interest, the desirededit becomes installed therein.

Any disease is conceivably treatable by such methods so long as deliveryto the appropriate cells is feasible. The person having ordinary skillin the art will be able to choose and/or select a PE deliverymethodology to suit the intended purpose and the intended target cells.

For example, in some embodiments, a method is provided that comprisesadministering to a subject having such a disease, e.g., a cancerassociated with a point mutation as described above, an effective amountof the prime editing system described herein that corrects the pointmutation or introduces a deactivating mutation into a disease-associatedgene as mediated by homology-directed repair in the presence of a donorDNA molecule comprising desired genetic change. In some embodiments, amethod is provided that comprises administering to a subject having sucha disease, e.g., a cancer associated with a point mutation as describedabove, an effective amount of the prime editing system described hereinthat corrects the point mutation or introduces a deactivating mutationinto a disease-associated gene. In some embodiments, the disease is aproliferative disease. In some embodiments, the disease is a geneticdisease. In some embodiments, the disease is a neoplastic disease. Insome embodiments, the disease is a metabolic disease. In someembodiments, the disease is a lysosomal storage disease. Other diseasesthat can be treated by correcting a point mutation or introducing adeactivating mutation into a disease-associated gene will be known tothose of skill in the art, and the disclosure is not limited in thisrespect.

The instant disclosure provides methods for the treatment of additionaldiseases or disorders, e.g., diseases or disorders that are associatedor caused by a point mutation that can be corrected by TPRT-mediatedgene editing. Some such diseases are described herein, and additionalsuitable diseases that can be treated with the strategies and fusionproteins provided herein will be apparent to those of skill in the artbased on the instant disclosure. Exemplary suitable diseases anddisorders are listed below. It will be understood that the numbering ofthe specific positions or residues in the respective sequences dependson the particular protein and numbering scheme used. Numbering might bedifferent, e.g., in precursors of a mature protein and the matureprotein itself, and differences in sequences from species to species mayaffect numbering. One of skill in the art will be able to identify therespective residue in any homologous protein and in the respectiveencoding nucleic acid by methods well known in the art, e.g., bysequence alignment and determination of homologous residues. Exemplarysuitable diseases and disorders include, without limitation:2-methyl-3-hydroxybutyric aciduria; 3 beta-Hydroxysteroid dehydrogenasedeficiency; 3-Methylglutaconic aciduria; 3-Oxo-5 alpha-steroid delta4-dehydrogenase deficiency; 46,XY sex reversal, type 1, 3, and 5;5-Oxoprolinase deficiency; 6-pyruvoyl-tetrahydropterin synthasedeficiency; Aarskog syndrome; Aase syndrome; Achondrogenesis type 2;Achromatopsia 2 and 7; Acquired long QT syndrome; Acrocallosal syndrome,Schinzel type; Acrocapitofemoral dysplasia; Acrodysostosis 2, with orwithout hormone resistance; Acroerythrokeratoderma; Acromicricdysplasia; Acth-independent macronodular adrenal hyperplasia 2;Activated PI3K-delta syndrome; Acute intermittent porphyria; deficiencyof Acyl-CoA dehydrogenase family, member 9; Adams-Oliver syndrome 5 and6; Adenine phosphoribosyltransferase deficiency; Adenylate kinasedeficiency; hemolytic anemia due to Adenylosuccinate lyase deficiency;Adolescent nephronophthisis; Renal-hepatic-pancreatic dysplasia; Meckelsyndrome type 7; Adrenoleukodystrophy; Adult junctional epidermolysisbullosa; Epidermolysis bullosa, junctional, localisata variant; Adultneuronal ceroid lipofuscinosis; Adult neuronal ceroid lipofuscinosis;Adult onset ataxia with oculomotor apraxia; ADULT syndrome;Afibrinogenemia and congenital Afibrinogenemia; autosomal recessiveAgammaglobulinemia 2; Age-related macular degeneration 3, 6, 11, and 12;Aicardi Goutieres syndromes 1, 4, and 5; Chilbain lupus 1; Alagillesyndromes 1 and 2; Alexander disease; Alkaptonuria; Allan-Herndon-Dudleysyndrome; Alopecia universalis congenital; Alpers encephalopathy;Alpha-1-antitrypsin deficiency; autosomal dominant, autosomal recessive,and X-linked recessive Alport syndromes; Alzheimer disease, familial, 3,with spastic paraparesis and apraxia; Alzheimer disease, types, 1, 3,and 4; hypocalcification type and hypomaturation type, IIA1 Amelogenesisimperfecta; Aminoacylase 1 deficiency; Amish infantile epilepsysyndrome; Amyloidogenic transthyretin amyloidosis; AmyloidCardiomyopathy, Transthyretin-related; Cardiomyopathy; Amyotrophiclateral sclerosis types 1, 6, 15 (with or without frontotemporaldementia), 22 (with or without frontotemporal dementia), and 10;Frontotemporal dementia with TDP43 inclusions, TARDBP-related; Andermannsyndrome; Andersen Tawil syndrome; Congenital long QT syndrome; Anemia,nonspherocytic hemolytic, due to G6PD deficiency; Angelman syndrome;Severe neonatal-onset encephalopathy with microcephaly; susceptibilityto Autism, X-linked 3; Angiopathy, hereditary, with nephropathy,aneurysms, and muscle cramps; Angiotensin i-converting enzyme, benignserum increase; Aniridia, cerebellar ataxia, and mental retardation;Anonychia; Antithrombin III deficiency; Antley-Bixler syndrome withgenital anomalies and disordered steroidogenesis; Aortic aneurysm,familial thoracic 4, 6, and 9; Thoracic aortic aneurysms and aorticdissections; Multisystemic smooth muscle dysfunction syndrome; Moyamoyadisease 5; Aplastic anemia; Apparent mineralocorticoid excess; Arginasedeficiency; Argininosuccinate lyase deficiency; Aromatase deficiency;Arrhythmogenic right ventricular cardiomyopathy types 5, 8, and 10;Primary familial hypertrophic cardiomyopathy; Arthrogryposis multiplexcongenita, distal, X-linked; Arthrogryposis renal dysfunctioncholestasis syndrome; Arthrogryposis, renal dysfunction, and cholestasis2; Asparagine synthetase deficiency; Abnormality of neuronal migration;Ataxia with vitamin E deficiency; Ataxia, sensory, autosomal dominant;Ataxia-telangiectasia syndrome; Hereditary cancer-predisposing syndrome;Atransferrinemia; Atrial fibrillation, familial, 11, 12, 13, and 16;Atrial septal defects 2, 4, and 7 (with or without atrioventricularconduction defects); Atrial standstill 2; Atrioventricular septal defect4; Atrophia bulborum hereditaria; ATR-X syndrome; Auriculocondylarsyndrome 2; Autoimmune disease, multisystem, infantile-onset; Autoimmunelymphoproliferative syndrome, type 1a; Autosomal dominant hypohidroticectodermal dysplasia; Autosomal dominant progressive externalophthalmoplegia with mitochondrial DNA deletions 1 and 3; Autosomaldominant torsion dystonia 4; Autosomal recessive centronuclear myopathy;Autosomal recessive congenital ichthyosis 1, 2, 3, 4A, and 4B; Autosomalrecessive cutis laxa type IA and 1B; Autosomal recessive hypohidroticectodermal dysplasia syndrome; Ectodermal dysplasia 11b;hypohidrotic/hair/tooth type, autosomal recessive; Autosomal recessivehypophosphatemic bone disease; Axenfeld-Rieger syndrome type 3;Bainbridge-Ropers syndrome; Bannayan-Riley-Ruvalcaba syndrome; PTENhamartoma tumor syndrome; Baraitser-Winter syndromes 1 and 2; Barakatsyndrome; Bardet-Biedl syndromes 1, 11, 16, and 19; Bare lymphocytesyndrome type 2, complementation group E; Bartter syndrome antenataltype 2; Bartter syndrome types 3, 3 with hypocalciuria, and 4; Basalganglia calcification, idiopathic, 4; Beaded hair; Benign familialhematuria; Benign familial neonatal seizures 1 and 2; Seizures, benignfamilial neonatal, 1, and/or myokymia; Seizures, Early infantileepileptic encephalopathy 7; Benign familial neonatal-infantile seizures;Benign hereditary chorea; Benign scapuloperoneal muscular dystrophy withcardiomyopathy; Bernard-Soulier syndrome, types A1 and A2 (autosomaldominant); Bestrophinopathy, autosomal recessive; beta Thalassemia;Bethlem myopathy and Bethlem myopathy 2; Bietti crystallinecorneoretinal dystrophy; Bile acid synthesis defect, congenital, 2;Biotinidase deficiency; Birk Barel mental retardation dysmorphismsyndrome; Blepharophimosis, ptosis, and epicanthus inversus; Bloomsyndrome; Borjeson-Forssman-Lehmann syndrome; Boucher Neuhausersyndrome; Brachydactyly types A1 and A2; Brachydactyly withhypertension; Brain small vessel disease with hemorrhage; Branched-chainketoacid dehydrogenase kinase deficiency; Branchiootic syndromes 2 and3; Breast cancer, early-onset; Breast-ovarian cancer, familial 1, 2, and4; Brittle cornea syndrome 2; Brody myopathy; Bronchiectasis with orwithout elevated sweat chloride 3; Brown-Vialetto-Van laere syndrome andBrown-Vialetto-Van Laere syndrome 2; Brugada syndrome; Brugada syndrome1; Ventricular fibrillation; Paroxysmal familial ventricularfibrillation; Brugada syndrome and Brugada syndrome 4; Long QT syndrome;Sudden cardiac death; Bull eye macular dystrophy; Stargardt disease 4;Cone-rod dystrophy 12; Bullous ichthyosiform erythroderma; Burn-Mckeownsyndrome; Candidiasis, familial, 2, 5, 6, and 8; Carbohydrate-deficientglycoprotein syndrome type I and II; Carbonic anhydrase VA deficiency,hyperammonemia due to; Carcinoma of colon; Cardiac arrhythmia; Long QTsyndrome, LQT1 subtype; Cardioencephalomyopathy, fatal infantile, due tocytochrome c oxidase deficiency; Cardiofaciocutaneous syndrome;Cardiomyopathy; Danon disease; Hypertrophic cardiomyopathy; Leftventricular noncompaction cardiomyopathy; Carnevale syndrome; Carneycomplex, type 1; Carnitine acylcamitine translocase deficiency;Carnitine palmitoyltransferase I, II, II (late onset), and II(infantile) deficiency; Cataract 1, 4, autosomal dominant, autosomaldominant, multiple types, with microcornea, coppock-like, juvenile, withmicrocomea and glucosuria, and nuclear diffuse nonprogressive;Catecholaminergic polymorphic ventricular tachycardia; Caudal regressionsyndrome; Cd8 deficiency, familial; Central core disease; Centromericinstability of chromosomes 1, 9 and 16 and immunodeficiency; Cerebellarataxia infantile with progressive external ophthalmoplegia andCerebellar ataxia, mental retardation, and dysequilibrium syndrome 2;Cerebral amyloid angiopathy, APP-related; Cerebral autosomal dominantand recessive arteriopathy with subcortical infarcts andleukoencephalopathy; Cerebral cavernous malformations 2;Cerebrooculofacioskeletal syndrome 2; Cerebro-oculo-facio-skeletalsyndrome; Cerebroretinal microangiopathy with calcifications and cysts;Ceroid lipofuscinosis neuronal 2, 6, 7, and 10; Ch\c3\xa9diak-Higashisyndrome, Chediak-Higashi syndrome, adult type; Charcot-Marie-Toothdisease types 1B, 2B2, 2C, 2F, 2I, 2U (axonal), 1C (demyelinating),dominant intermediate C, recessive intermediate A, 2A2, 4C, 4D, 4H, IF,IVF, and X; Scapuloperoneal spinal muscular atrophy; Distal spinalmuscular atrophy, congenital nonprogressive; Spinal muscular atrophy,distal, autosomal recessive, 5; CHARGE association; Childhoodhypophosphatasia; Adult hypophosphatasia; Cholecystitis; Progressivefamilial intrahepatic cholestasis 3; Cholestasis, intrahepatic, ofpregnancy 3; Cholestanol storage disease; Cholesterol monooxygenase(side-chain cleaving) deficiency; Chondrodysplasia Blomstrand type;Chondrodysplasia punctata 1, X-linked recessive and 2 X-linked dominant;CHOPS syndrome; Chronic granulomatous disease, autosomal recessivecytochrome b-positive, types 1 and 2; Chudley-McCullough syndrome;Ciliary dyskinesia, primary, 7, 11, 15, 20 and 22; Citrullinemia type I;Citrullinemia type I and II; Cleidocranial dysostosis; C-like syndrome;Cockayne syndrome type A; Coenzyme Q10 deficiency, primary 1, 4, and 7;Coffin Siris/Intellectual Disability; Coffin-Lowry syndrome; Cohensyndrome; Cold-induced sweating syndrome 1; COLE-CARPENTER SYNDROME 2;Combined cellular and humoral immune defects with granulomas; Combinedd-2- and 1-2-hydroxyglutaric aciduria; Combined malonic andmethylmalonic aciduria; Combined oxidative phosphorylation deficiencies1, 3, 4, 12, 15, and 25; Combined partial and complete17-alpha-hydroxylase/17,20-lyase deficiency; Common variableimmunodeficiency 9; Complement component 4, partial deficiency of, dueto dysfunctional cl inhibitor; Complement factor B deficiency; Conemonochromatism; Cone-rod dystrophy 2 and 6; Cone-rod dystrophyamelogenesis imperfecta; Congenital adrenal hyperplasia and Congenitaladrenal hypoplasia, X-linked; Congenital amegakaryocyticthrombocytopenia; Congenital aniridia; Congenital centralhypoventilation; Hirschsprung disease 3; Congenital contracturalarachnodactyly; Congenital contractures of the limbs and face,hypotonia, and developmental delay; Congenital disorder of glycosylationtypes 1B, 1D, 1G, 1H, 1J, 1K, 1N, 1P, 2C, 2J, 2K, IIm; Congenitaldyserythropoietic anemia, type I and II; Congenital ectodermal dysplasiaof face; Congenital erythropoietic porphyria; Congenital generalizedlipodystrophy type 2; Congenital heart disease, multiple types, 2;Congenital heart disease; Interrupted aortic arch; Congenital lipomatousovergrowth, vascular malformations, and epidermal nevi; Non-small celllung cancer; Neoplasm of ovary; Cardiac conduction defect, nonspecific;Congenital microvillous atrophy; Congenital muscular dystrophy;Congenital muscular dystrophy due to partial LAMA2 deficiency;Congenital muscular dystrophy-dystroglycanopathy with brain and eyeanomalies, types A2, A7, A8, A11, and A14; Congenital musculardystrophy-dystroglycanopathy with mental retardation, types B2, B3, B5,and B15; Congenital muscular dystrophy-dystroglycanopathy without mentalretardation, type B5; Congenital muscular hypertrophy-cerebral syndrome;Congenital myasthenic syndrome, acetazolamide-responsive; Congenitalmyopathy with fiber type disproportion; Congenital ocular coloboma;Congenital stationary night blindness, type 1A, 1B, 1C, 1E, 1F, and 2A;Coproporphyria; Cornea plana 2; Corneal dystrophy, Fuchs endothelial, 4;Corneal endothelial dystrophy type 2; Corneal fragility keratoglobus,blue sclerae and joint hypermobility; Cornelia de Lange syndromes 1 and5; Coronary artery disease, autosomal dominant 2; Coronary heartdisease; Hyperalphalipoproteinemia 2; Cortical dysplasia, complex, withother brain malformations 5 and 6; Cortical malformations, occipital;Corticosteroid-binding globulin deficiency; Corticosterone methyloxidasetype 2 deficiency; Costello syndrome; Cowden syndrome 1; Coxa plana;Craniodiaphyseal dysplasia, autosomal dominant; Craniosynostosis 1 and4; Craniosynostosis and dental anomalies; Creatine deficiency, X-linked;Crouzon syndrome; Cryptophthalmos syndrome; Cryptorchidism, unilateralor bilateral; Cushing symphalangism; Cutaneous malignant melanoma 1;Cutis laxa with osteodystrophy and with severe pulmonary,gastrointestinal, and urinary abnormalities; Cyanosis, transientneonatal and atypical nephropathic; Cystic fibrosis; Cystinuria;Cytochrome c oxidase i deficiency; Cytochrome-c oxidase deficiency;D-2-hydroxyglutaric aciduria 2; Darier disease, segmental; Deafness withlabyrinthine aplasia microtia and microdontia (LAMM); Deafness,autosomal dominant 3a, 4, 12, 13, 15, autosomal dominant nonsyndromicsensorineural 17, 20, and 65; Deafness, autosomal recessive IA, 2, 3, 6,8, 9, 12, 15, 16, 18b, 22, 28, 31, 44, 49, 63, 77, 86, and 89; Deafness,cochlear, with myopia and intellectual impairment, without vestibularinvolvement, autosomal dominant, X-linked 2; Deficiency of2-methylbutyryl-CoA dehydrogenase; Deficiency of 3-hydroxyacyl-CoAdehydrogenase; Deficiency of alpha-mannosidase; Deficiency ofaromatic-L-amino-acid decarboxylase; Deficiency of bisphosphoglyceratemutase; Deficiency of butyryl-CoA dehydrogenase; Deficiency offerroxidase; Deficiency of galactokinase; Deficiency of guanidinoacetatemethyltransferase; Deficiency of hyaluronoglucosaminidase; Deficiency ofribose-5-phosphate isomerase; Deficiency of steroid11-beta-monooxygenase; Deficiency of UDPglucose-hexose-1-phosphateuridylyltransferase; Deficiency of xanthine oxidase; Dejerine-Sottasdisease; Charcot-Marie-Tooth disease, types ID and IVF; Dejerine-Sottassyndrome, autosomal dominant; Dendritic cell, monocyte, B lymphocyte,and natural killer lymphocyte deficiency; Desbuquois dysplasia 2;Desbuquois syndrome; DFNA 2 Nonsyndromic Hearing Loss; Diabetes mellitusand insipidus with optic atrophy and deafness; Diabetes mellitus, type2, and insulin-dependent, 20; Diamond-Blackfan anemia 1, 5, 8, and 10;Diarrhea 3 (secretory sodium, congenital, syndromic) and 5 (with tuftingenteropathy, congenital); Dicarboxylic aminoaciduria; Diffusepalmoplantar keratoderma, Bothnian type; Digitorenocerebral syndrome;Dihydropteridine reductase deficiency; Dilated cardiomyopathy 1A, 1AA,1C, 1G, 1BB, 1DD, 1FF, 1HH, 1I, 1KK, 1N, 1S, 1Y, and 3B; Leftventricular noncompaction 3; Disordered steroidogenesis due tocytochrome p450 oxidoreductase deficiency; Distal arthrogryposis type2B; Distal hereditary motor neuronopathy type 2B; Distal myopathyMarkesbery-Griggs type; Distal spinal muscular atrophy, X-linked 3;Distichiasis-lymphedema syndrome; Dominant dystrophic epidermolysisbullosa with absence of skin; Dominant hereditary optic atrophy; DonnaiBarrow syndrome; Dopamine beta hydroxylase deficiency; Dopamine receptord2, reduced brain density of; Dowling-degos disease 4; Doyne honeycombretinal dystrophy; Malattia leventinese; Duane syndrome type 2;Dubin-Johnson syndrome; Duchenne muscular dystrophy; Becker musculardystrophy; Dysfibrinogenemia; Dyskeratosis congenita autosomal dominantand autosomal dominant, 3; Dyskeratosis congenita, autosomal recessive,1, 3, 4, and 5; Dyskeratosis congenita X-linked; Dyskinesia, familial,with facial myokymia; Dysplasminogenemia; Dystonia 2 (torsion, autosomalrecessive), 3 (torsion, X-linked), 5 (Dopa-responsive type), 10, 12, 16,25, 26 (Myoclonic); Seizures, benign familial infantile, 2; Earlyinfantile epileptic encephalopathy 2, 4, 7, 9, 10, 11, 13, and 14;Atypical Rett syndrome; Early T cell progenitor acute lymphoblasticleukemia; Ectodermal dysplasia skin fragility syndrome; Ectodermaldysplasia-syndactyly syndrome 1; Ectopia lentis, isolated autosomalrecessive and dominant; Ectrodactyly, ectodermal dysplasia, and cleftlip/palate syndrome 3; Ehlers-Danlos syndrome type 7 (autosomalrecessive), classic type, type 2 (progeroid), hydroxylysine-deficient,type 4, type 4 variant, and due to tenascin-X deficiency; Eichsfeld typecongenital muscular dystrophy; Endocrine-cerebroosteodysplasia; Enhanceds-cone syndrome; Enlarged vestibular aqueduct syndrome; Enterokinasedeficiency; Epidermodysplasia verruciformis; Epidermolysa bullosasimplex and limb girdle muscular dystrophy, simplex with mottledpigmentation, simplex with pyloric atresia, simplex, autosomalrecessive, and with pyloric atresia; Epidermolytic palmoplantarkeratoderma; Familial febrile seizures 8; Epilepsy, childhood absence 2,12 (idiopathic generalized, susceptibility to) 5 (nocturnal frontallobe), nocturnal frontal lobe type 1, partial, with variable foci,progressive myoclonic 3, and X-linked, with variable learningdisabilities and behavior disorders; Epileptic encephalopathy,childhood-onset, early infantile, 1, 19, 23, 25, 30, and 32; Epiphysealdysplasia, multiple, with myopia and conductive deafness; Episodicataxia type 2; Episodic pain syndrome, familial, 3; Epstein syndrome;Fechtner syndrome; Erythropoietic protoporphyria; Estrogen resistance;Exudative vitreoretinopathy 6; Fabry disease and Fabry disease, cardiacvariant; Factor H, VII, X, v and factor viii, combined deficiency of 2,xiii, a subunit, deficiency; Familial adenomatous polyposis 1 and 3;Familial amyloid nephropathy with urticaria and deafness; Familial coldurticarial; Familial aplasia of the vermis; Familial benign pemphigus;Familial cancer of breast; Breast cancer, susceptibility to;Osteosarcoma; Pancreatic cancer 3; Familial cardiomyopathy; Familialcold autoinflammatory syndrome 2; Familial colorectal cancer; Familialexudative vitreoretinopathy, X-linked; Familial hemiplegic migrainetypes 1 and 2; Familial hypercholesterolemia; Familial hypertrophiccardiomyopathy 1, 2, 3, 4, 7, 10, 23 and 24; Familialhypokalemia-hypomagnesemia; Familial hypoplastic, glomerulocystickidney; Familial infantile myasthenia; Familial juvenile gout; FamilialMediterranean fever and Familial mediterranean fever, autosomaldominant; Familial porencephaly; Familial porphyria cutanea tarda;Familial pulmonary capillary hemangiomatosis; Familial renal glucosuria;Familial renal hypouricemia; Familial restrictive cardiomyopathy 1;Familial type 1 and 3 hyperlipoproteinemia; Fanconi anemia,complementation group E, I, N, and O; Fanconi-Bickel syndrome; Favism,susceptibility to; Febrile seizures, familial, 11; Feingold syndrome 1;Fetal hemoglobin quantitative trait locus 1; FG syndrome and FG syndrome4; Fibrosis of extraocular muscles, congenital, 1, 2, 3a (with orwithout extraocular involvement), 3b; Fish-eye disease; Fleck cornealdystrophy; Floating-Harbor syndrome; Focal epilepsy with speech disorderwith or without mental retardation; Focal segmental glomerulosclerosis5; Forebrain defects; Frank Ter Haar syndrome; Borrone Di Rocco Crovatosyndrome; Frasier syndrome; Wilms tumor 1; Freeman-Sheldon syndrome;Frontometaphyseal dysplasia land 3; Frontotemporal dementia;Frontotemporal dementia and/or amyotrophic lateral sclerosis 3 and 4;Frontotemporal Dementia Chromosome 3-Linked and Frontotemporal dementiaubiquitin-positive; Fructose-biphosphatase deficiency; Fuhrmannsyndrome; Gamma-aminobutyric acid transaminase deficiency;Gamstorp-Wohlfart syndrome; Gaucher disease type 1 and Subacuteneuronopathic; Gaze palsy, familial horizontal, with progressivescoliosis; Generalized dominant dystrophic epidermolysis bullosa;Generalized epilepsy with febrile seizures plus 3, type 1, type 2;Epileptic encephalopathy Lennox-Gastaut type; Giant axonal neuropathy;Glanzmann thrombasthenia; Glaucoma 1, open angle, e, F, and G; Glaucoma3, primary congenital, d; Glaucoma, congenital and Glaucoma, congenital,Coloboma; Glaucoma, primary open angle, juvenile-onset; Gliomasusceptibility 1; Glucose transporter type 1 deficiency syndrome;Glucose-6-phosphate transport defect; GLUT1 deficiency syndrome 2;Epilepsy, idiopathic generalized, susceptibility to, 12; Glutamateformiminotransferase deficiency; Glutaric acidemia IIA and IIB; Glutaricaciduria, type 1; Gluthathione synthetase deficiency; Glycogen storagedisease 0 (muscle), II (adult form), IXa2, IXc, type IA; type II, typeIV, IV (combined hepatic and myopathic), type V, and type VI;Goldmann-Favre syndrome; Gordon syndrome; Gorlin syndrome;Holoprosencephaly sequence; Holoprosencephaly 7; Granulomatous disease,chronic, X-linked, variant; Granulosa cell tumor of the ovary; Grayplatelet syndrome; Griscelli syndrome type 3; Groenouw corneal dystrophytype I; Growth and mental retardation, mandibulofacial dysostosis,microcephaly, and cleft palate; Growth hormone deficiency with pituitaryanomalies; Growth hormone insensitivity with immunodeficiency; GTPcyclohydrolase I deficiency; Hajdu-Cheney syndrome; Hand foot uterussyndrome; Hearing impairment; Hemangioma, capillary infantile;Hematologic neoplasm; Hemochromatosis type 1, 2B, and 3; Microvascularcomplications of diabetes 7; Transferrin serum level quantitative traitlocus 2; Hemoglobin H disease, nondeletional; Hemolytic anemia,nonspherocytic, due to glucose phosphate isomerase deficiency;Hemophagocytic lymphohistiocytosis, familial, 2; Hemophagocyticlymphohistiocytosis, familial, 3; Heparin cofactor II deficiency;Hereditary acrodermatitis enteropathica; Hereditary breast and ovariancancer syndrome; Ataxia-telangiectasia-like disorder; Hereditary diffusegastric cancer; Hereditary diffuse leukoencephalopathy with spheroids;Hereditary factors II, IX, VIII deficiency disease; Hereditaryhemorrhagic telangiectasia type 2; Hereditary insensitivity to pain withanhidrosis; Hereditary lymphedema type I; Hereditary motor and sensoryneuropathy with optic atrophy; Hereditary myopathy with earlyrespiratory failure; Hereditary neuralgic amyotrophy; HereditaryNonpolyposis Colorectal Neoplasms; Lynch syndrome I and II; Hereditarypancreatitis; Pancreatitis, chronic, susceptibility to; Hereditarysensory and autonomic neuropathy type IIB and IIA; Hereditarysideroblastic anemia; Hermansky-Pudlak syndrome 1, 3, 4, and 6;Heterotaxy, visceral, 2, 4, and 6, autosomal; Heterotaxy, visceral,X-linked; Heterotopia; Histiocytic medullary reticulosis;Histiocytosis-lymphadenopathy plus syndrome; Holocarboxylase synthetasedeficiency; Holoprosencephaly 2, 3,7, and 9; Holt-Oram syndrome;Homocysteinemia due to MTHFR deficiency, CBS deficiency, andHomocystinuria, pyridoxine-responsive; Homocystinuria-Megaloblasticanemia due to defect in cobalamin metabolism, cblE complementation type;Howel-Evans syndrome; Hurler syndrome; Hutchinson-Gilford syndrome;Hydrocephalus; Hyperammonemia, type III; Hypercholesterolaemia andHypercholesterolemia, autosomal recessive; Hyperekplexia 2 andHyperekplexia hereditary; Hyperferritinemia cataract syndrome;Hyperglycinuria; Hyperimmunoglobulin D with periodic fever; Mevalonicaciduria; Hyperimmunoglobulin E syndrome; Hyperinsulinemic hypoglycemiafamilial 3, 4, and 5; Hyperinsulinism-hyperammonemia syndrome;Hyperlysinemia; Hypermanganesemia with dystonia, polycythemia andcirrhosis; Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome;Hyperparathyroidism 1 and 2; Hyperparathyroidism, neonatal severe;Hyperphenylalaninemia, bh4-deficient, a, due to partial pts deficiency,BH4-deficient, D, and non-pku; Hyperphosphatasia with mental retardationsyndrome 2, 3, and 4; Hypertrichotic osteochondrodysplasia;Hypobetalipoproteinemia, familial, associated with apob32; Hypocalcemia,autosomal dominant 1; Hypocalciuric hypercalcemia, familial, types 1 and3; Hypochondrogenesis; Hypochromic microcytic anemia with iron overload;Hypoglycemia with deficiency of glycogen synthetase in the liver;Hypogonadotropic hypogonadism 11 with or without anosmia; Hypohidroticectodermal dysplasia with immune deficiency; Hypohidrotic X-linkedectodermal dysplasia; Hypokalemic periodic paralysis 1 and 2;Hypomagnesemia 1, intestinal; Hypomagnesemia, seizures, and mentalretardation; Hypomyelinating leukodystrophy 7; Hypoplastic left heartsyndrome; Atrioventricular septal defect and common atrioventricularjunction; Hypospadias 1 and 2, X-linked; Hypothyroidism, congenital,nongoitrous, 1; Hypotrichosis 8 and 12;Hypotrichosis-lymphedema-telangiectasia syndrome; I blood group system;Ichthyosis bullosa of Siemens; Ichthyosis exfoliativa; Ichthyosisprematurity syndrome; Idiopathic basal ganglia calcification 5;Idiopathic fibrosing alveolitis, chronic form; Dyskeratosis congenita,autosomal dominant, 2 and 5; Idiopathic hypercalcemia of infancy; Immunedysfunction with T-cell inactivation due to calcium entry defect 2;Immunodeficiency 15, 16, 19, 30, 31C, 38, 40, 8, due to defect incd3-zeta, with hyper IgM type 1 and 2, and X-Linked, with magnesiumdefect, Epstein-Barr virus infection, and neoplasia;Immunodeficiency-centromeric instability-facial anomalies syndrome 2;Inclusion body myopathy 2 and 3; Nonaka myopathy; Infantile convulsionsand paroxysmal choreoathetosis, familial; Infantile corticalhyperostosis; Infantile GM1 gangliosidosis; Infantile hypophosphatasia;Infantile nephronophthisis; Infantile nystagmus, X-linked; InfantileParkinsonism-dystonia; Infertility associated with multi-tailedspermatozoa and excessive DNA; Insulin resistance; Insulin-resistantdiabetes mellitus and acanthosis nigricans; Insulin-dependent diabetesmellitus secretory diarrhea syndrome; Interstitial nephritis,karyomegalic; Intrauterine growth retardation, metaphyseal dysplasia,adrenal hypoplasia congenita, and genital anomalies; Iodotyrosylcoupling defect; IRAK4 deficiency; Iridogoniodysgenesis dominant typeand type 1; Iron accumulation in brain; Ischiopatellar dysplasia; Isletcell hyperplasia; Isolated 17,20-lyase deficiency; Isolated lutropindeficiency; Isovaleryl-CoA dehydrogenase deficiency; Jankovic Riverasyndrome; Jervell and Lange-Nielsen syndrome 2; Joubert syndrome 1, 6,7, 9/15 (digenic), 14, 16, and 17, and Orofaciodigital syndrome xiv;Junctional epidermolysis bullosa gravis of Herlitz; JuvenileGM>1<gangliosidosis; Juvenile polyposis syndrome; Juvenilepolyposis/hereditary hemorrhagic telangiectasia syndrome; Juvenileretinoschisis; Kabuki make-up syndrome; Kallmann syndrome 1, 2, and 6;Delayed puberty; Kanzaki disease; Karak syndrome; Kartagener syndrome;Kenny-Caffey syndrome type 2; Keppen-Lubinsky syndrome; Keratoconus 1;Keratosis follicularis; Keratosis palmoplantaris striata 1; Kindlersyndrome; L-2-hydroxyglutaric aciduria; Larsen syndrome, dominant type;Lattice corneal dystrophy Type III; Leber amaurosis; Zellweger syndrome;Peroxisome biogenesis disorders; Zellweger syndrome spectrum; Lebercongenital amaurosis 11, 12, 13, 16, 4, 7, and 9; Leber optic atrophy;Aminoglycoside-induced deafness; Deafness, nonsyndromic sensorineural,mitochondrial; Left ventricular noncompaction 5; Left-right axismalformations; Leigh disease; Mitochondrial short-chain Enoyl-CoAHydratase 1 deficiency; Leigh syndrome due to mitochondrial complex Ideficiency; Leiner disease; Leri Weill dyschondrosteosis; Lethalcongenital contracture syndrome 6; Leukocyte adhesion deficiency type Iand III; Leukodystrophy, Hypomyelinating, 11 and 6; Leukoencephalopathywith ataxia, with Brainstem and Spinal Cord Involvement and LactateElevation, with vanishing white matter, and progressive, with ovarianfailure; Leukonychia totalis; Lewy body dementia; Lichtenstein-KnorrSyndrome; Li-Fraumeni syndrome 1; Lig4 syndrome; Limb-girdle musculardystrophy, type 1B, 2A, 2B, 2D, C1, C5, C9, C14; Congenital musculardystrophy-dystroglycanopathy with brain and eye anomalies, type A14 andB14; Lipase deficiency combined; Lipid proteinosis; Lipodystrophy,familial partial, type 2 and 3; Lissencephaly 1, 2 (X-linked), 3, 6(with microcephaly), X-linked; Subcortical laminar heterotopia,X-linked; Liver failure acute infantile; Loeys-Dietz syndrome 1, 2, 3;Long QT syndrome 1, 2, 2/9, 2/5, (digenic), 3, 5 and 5, acquired,susceptibility to; Lung cancer; Lymphedema, hereditary, id; Lymphedema,primary, with myelodysplasia; Lymphoproliferative syndrome 1, 1(X-linked), and 2; Lysosomal acid lipase deficiency; Macrocephaly,macrosomia, facial dysmorphism syndrome; Macular dystrophy, vitelliform,adult-onset; Malignant hyperthermia susceptibility type 1; Malignantlymphoma, non-Hodgkin; Malignant melanoma; Malignant tumor of prostate;Mandibuloacral dysostosis; Mandibuloacral dysplasia with type A or Blipodystrophy, atypical; Mandibulofacial dysostosis, Treacher Collinstype, autosomal recessive; Mannose-binding protein deficiency; Maplesyrup urine disease type IA and type 3; Marden Walker like syndrome;Marfan syndrome; Marinesco-Sj\xc3\xb6gren syndrome; Martsolf syndrome;Maturity-onset diabetes of the young, type 1, type 2, type 11, type 3,and type 9; May-Hegglin anomaly; MYH9 related disorders; Sebastiansyndrome; McCune-Albright syndrome; Somatotroph adenoma; Sexcord-stromal tumor; Cushing syndrome; McKusick Kaufman syndrome; McLeodneuroacanthocytosis syndrome; Meckel-Gruber syndrome; Medium-chainacyl-coenzyme A dehydrogenase deficiency; Medulloblastoma;Megalencephalic leukoencephalopathy with subcortical cysts land 2a;Megalencephaly cutis marmorata telangiectatica congenital; PIK3CARelated Overgrowth Spectrum;Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2;Megaloblastic anemia, thiamine-responsive, with diabetes mellitus andsensorineural deafness; Meier-Gorlin syndromes land 4; Melnick-Needlessyndrome; Meningioma; Mental retardation, X-linked, 3, 21, 30, and 72;Mental retardation and microcephaly with pontine and cerebellarhypoplasia; Mental retardation X-linked syndromic 5; Mental retardation,anterior maxillary protrusion, and strabismus; Mental retardation,autosomal dominant 12, 13, 15, 24, 3, 30, 4, 5, 6,and 9; Mentalretardation, autosomal recessive 15, 44, 46, and 5; Mental retardation,stereotypic movements, epilepsy, and/or cerebral malformations; Mentalretardation, syndromic, Claes-Jensen type, X-linked; Mental retardation,X-linked, nonspecific, syndromic, Hedera type, and syndromic, wu type;Merosin deficient congenital muscular dystrophy; Metachromaticleukodystrophy juvenile, late infantile, and adult types; Metachromaticleukodystrophy; Metatrophic dysplasia; Methemoglobinemia types I and 2;Methionine adenosyltransferase deficiency, autosomal dominant;Methylmalonic acidemia with homocystinuria; Methylmalonic aciduria cblBtype; Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency;METHYLMALONIC ACIDURIA, mut(0) TYPE; Microcephalic osteodysplasticprimordial dwarfism type 2; Microcephaly with or withoutchorioretinopathy, lymphedema, or mental retardation; Microcephaly,hiatal hernia and nephrotic syndrome; Microcephaly; Hypoplasia of thecorpus callosum; Spastic paraplegia 50, autosomal recessive; Globaldevelopmental delay; CNS hypomyelination; Brain atrophy; Microcephaly,normal intelligence and immunodeficiency; Microcephaly-capillarymalformation syndrome; Microcytic anemia; Microphthalmia syndromic 5, 7,and 9; Microphthalmia, isolated 3, 5, 6, 8, and with coloboma 6;Microspherophakia; Migraine, familial basilar; Miller syndrome; Minicoremyopathy with external ophthalmoplegia; Myopathy, congenital with cores;Mitchell-Riley syndrome; mitochondrial 3-hydroxy-3-methylglutaryl-CoAsynthase deficiency; Mitochondrial complex I, II, III, III (nuclear type2, 4, or 8) deficiency; Mitochondrial DNA depletion syndrome 11, 12(cardiomyopathic type), 2, 4B (MNGIE type), 8B (MNGIE type);Mitochondrial DNA-depletion syndrome 3 and 7, hepatocerebral types, and13 (encephalomyopathic type); Mitochondrial phosphate carrier andpyruvate carrier deficiency; Mitochondrial trifunctional proteindeficiency; Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency;Miyoshi muscular dystrophy 1; Myopathy, distal, with anterior tibialonset; Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency,complementation group A; Mowat-Wilson syndrome; Mucolipidosis III Gamma;Mucopolysaccharidosis type VI, type VI (severe), and type VII;Mucopolysaccharidosis, MPS-I-H/S, MPS-II, MPS-III-A, MPS-II-B,MPS-III-C, MPS-IV-A, MPS-IV-B; Retinitis Pigmentosa 73; GangliosidosisGM1 type1 (with cardiac involvement) 3; Multicentric osteolysisnephropathy; Multicentric osteolysis, nodulosis and arthropathy;Multiple congenital anomalies; Atrial septal defect 2; Multiplecongenital anomalies-hypotonia-seizures syndrome 3; Multiple Cutaneousand Mucosal Venous Malformations; Multiple endocrine neoplasia, typesland 4; Multiple epiphyseal dysplasia 5 or Dominant; Multiplegastrointestinal atresias; Multiple pterygium syndrome Escobar type;Multiple sulfatase deficiency; Multiple synostoses syndrome 3; MuscleAMP guanine oxidase deficiency; Muscle eye brain disease; Musculardystrophy, congenital, megaconial type; Myasthenia, familial infantile,1; Myasthenic Syndrome, Congenital, 11, associated with acetylcholinereceptor deficiency; Myasthenic Syndrome, Congenital, 17, 2A(slow-channel), 4B (fast-channel), and without tubular aggregates;Myeloperoxidase deficiency; MYH-associated polyposis; Endometrialcarcinoma; Myocardial infarction 1; Myoclonic dystonia; Myoclonic-AtonicEpilepsy; Myoclonus with epilepsy with ragged red fibers; Myofibrillarmyopathy 1 and ZASP-related; Myoglobinuria, acute recurrent, autosomalrecessive; Myoneural gastrointestinal encephalopathy syndrome;Cerebellar ataxia infantile with progressive external ophthalmoplegia;Mitochondrial DNA depletion syndrome 4B, MNGIE type; Myopathy,centronuclear, 1, congenital, with excess of muscle spindles, distal, 1,lactic acidosis, and sideroblastic anemia 1, mitochondrial progressivewith congenital cataract, hearing loss, and developmental delay, andtubular aggregate, 2; Myopia 6; Myosclerosis, autosomal recessive;Myotonia congenital; Congenital myotonia, autosomal dominant andrecessive forms; Nail-patella syndrome; Nance-Horan syndrome;Nanophthalmos 2; Navajo neurohepatopathy; Nemaline myopathy 3 and 9;Neonatal hypotonia; Intellectual disability; Seizures; Delayed speechand language development; Mental retardation, autosomal dominant 31;Neonatal intrahepatic cholestasis caused by citrin deficiency;Nephrogenic diabetes insipidus, Nephrogenic diabetes insipidus,X-linked; Nephrolithiasis/osteoporosis, hypophosphatemic, 2;Nephronophthisis 13, 15 and 4; Infertility; Cerebello-oculo-renalsyndrome (nephronophthisis, oculomotor apraxia and cerebellarabnormalities); Nephrotic syndrome, type 3, type 5, with or withoutocular abnormalities, type 7, and type 9; Nestor-Guillermo progeriasyndrome; Neu-Laxova syndrome 1; Neurodegeneration with brain ironaccumulation 4 and 6; Neuroferritinopathy; Neurofibromatosis, type landtype 2; Neurofibrosarcoma; Neurohypophyseal diabetes insipidus;Neuropathy, Hereditary Sensory, Type IC; Neutral 1 amino acid transportdefect; Neutral lipid storage disease with myopathy; Neutrophilimmunodeficiency syndrome; Nicolaides-Baraitser syndrome; Niemann-Pickdisease type C1, C2, type A, and type C1, adult form; Non-ketotichyperglycinemia; Noonan syndrome 1 and 4, LEOPARD syndrome 1; Noonansyndrome-like disorder with or without juvenile myelomonocytic leukemia;Normokalemic periodic paralysis, potassium-sensitive; Norum disease;Epilepsy, Hearing Loss, And Mental Retardation Syndrome; MentalRetardation, X-Linked 102 and syndromic 13; Obesity; Ocular albinism,type I; Oculocutaneous albinism type 1B, type 3, and type 4;Oculodentodigital dysplasia; Odontohypophosphatasia; Odontotrichomelicsyndrome; Oguchi disease; Oligodontia-colorectal cancer syndrome; OpitzG/BBB syndrome; Optic atrophy 9; Oral-facial-digital syndrome; Ornithineaminotransferase deficiency; Orofacial cleft 11 and 7, Cleftlip/palate-ectodermal dysplasia syndrome; Orstavik Lindemann Solbergsyndrome; Osteoarthritis with mild chondrodysplasia; Osteochondritisdissecans; Osteogenesis imperfecta type 12, type 5, type 7, type 8, typeI, type III, with normal sclerae, dominant form, recessive perinatallethal; Osteopathia striata with cranial sclerosis; Osteopetrosisautosomal dominant type 1 and 2, recessive 4, recessive 1, recessive 6;Osteoporosis with pseudoglioma; Oto-palato-digital syndrome, types I andII; Ovarian dysgenesis 1; Ovarioleukodystrophy; Pachyonychia congenita 4and type 2; Paget disease of bone, familial; Pallister-Hall syndrome;Palmoplantar keratoderma, nonepidermolytic, focal or diffuse; Pancreaticagenesis and congenital heart disease; Papillon-Lef\xc3\xa8vre syndrome;Paragangliomas 3; Paramyotonia congenita of von Eulenburg; Parathyroidcarcinoma; Parkinson disease 14, 15, 19 (juvenile-onset), 2, 20(early-onset), 6, (autosomal recessive early-onset, and 9; Partialalbinism; Partial hypoxanthine-guanine phosphoribosyltransferasedeficiency; Patterned dystrophy of retinal pigment epithelium; PC-K6a;Pelizaeus-Merzbacher disease; Pendred syndrome; Peripheral demyelinatingneuropathy, central dysmyelination; Hirschsprung disease; Permanentneonatal diabetes mellitus; Diabetes mellitus, permanent neonatal, withneurologic features; Neonatal insulin-dependent diabetes mellitus;Maturity-onset diabetes of the young, type 2; Peroxisome biogenesisdisorder 14B, 2A, 4A, 5B, 6A, 7A, and 7B; Perrault syndrome 4; Perrysyndrome; Persistent hyperinsulinemic hypoglycemia of infancy; familialhyperinsulinism; Phenotypes; Phenylketonuria; Pheochromocytoma;Hereditary Paraganglioma-Pheochromocytoma Syndromes; Paragangliomas 1;Carcinoid tumor of intestine; Cowden syndrome 3; Phosphoglyceratedehydrogenase deficiency; Phosphoglycerate kinase 1 deficiency;Photosensitive trichothiodystrophy; Phytanic acid storage disease; Pickdisease; Pierson syndrome; Pigmentary retinal dystrophy; Pigmentednodular adrenocortical disease, primary, 1; Pilomatrixoma; Pitt-Hopkinssyndrome; Pituitary dependent hypercortisolism; Pituitary hormonedeficiency, combined 1, 2, 3, and 4; Plasminogen activator inhibitortype 1 deficiency; Plasminogen deficiency, type I; Platelet-typebleeding disorder 15 and 8; Poikiloderma, hereditary fibrosing, withtendon contractures, myopathy, and pulmonary fibrosis; Polycystic kidneydisease 2, adult type, and infantile type; Polycystic lipomembranousosteodysplasia with sclerosing leukoencephalopathy; Polyglucosan bodymyopathy 1 with or without immunodeficiency; Polymicrogyria, asymmetric,bilateral frontoparietal; Polyneuropathy, hearing loss, ataxia,retinitis pigmentosa, and cataract; Pontocerebellar hypoplasia type 4;Popliteal pterygium syndrome; Porencephaly 2; Porokeratosis 8,disseminated superficial actinic type; Porphobilinogen synthasedeficiency; Porphyria cutanea tarda; Posterior column ataxia withretinitis pigmentosa; Posterior polar cataract type 2; Prader-Willi-likesyndrome; Premature ovarian failure 4, 5, 7, and 9; Primary autosomalrecessive microcephaly 10, 2, 3, and 5; Primary ciliary dyskinesia 24;Primary dilated cardiomyopathy; Left ventricular noncompaction 6; 4,Left ventricular noncompaction 10; Paroxysmal atrial fibrillation;Primary hyperoxaluria, type I, type, and type III; Primary hypertrophicosteoarthropathy, autosomal recessive 2; Primary hypomagnesemia; Primaryopen angle glaucoma juvenile onset 1; Primary pulmonary hypertension;Primrose syndrome; Progressive familial heart block type 1B; Progressivefamilial intrahepatic cholestasis 2 and 3; Progressive intrahepaticcholestasis; Progressive myoclonus epilepsy with ataxia; Progressivepseudorheumatoid dysplasia; Progressive sclerosing poliodystrophy;Prolidase deficiency; Proline dehydrogenase deficiency; Schizophrenia 4;Properdin deficiency, X-linked; Propionic academia; Proproteinconvertase 1/3 deficiency; Prostate cancer, hereditary, 2; Protandefect; Proteinuria; Finnish congenital nephrotic syndrome; Proteussyndrome; Breast adenocarcinoma; Pseudoachondroplasticspondyloepiphyseal dysplasia syndrome; Pseudohypoaldosteronism type 1autosomal dominant and recessive and type 2; Pseudohypoparathyroidismtype 1A, Pseudopseudohypoparathyroidism; Pseudoneonataladrenoleukodystrophy; Pseudoprimary hyperaldosteronism; Pseudoxanthomaelasticum; Generalized arterial calcification of infancy 2;Pseudoxanthoma elasticum-like disorder with multiple coagulation factordeficiency; Psoriasis susceptibility 2; PTEN hamartoma tumor syndrome;Pulmonary arterial hypertension related to hereditary hemorrhagictelangiectasia; Pulmonary Fibrosis And/Or Bone Marrow Failure,Telomere-Related, 1 and 3; Pulmonary hypertension, primary, 1, withhereditary hemorrhagic telangiectasia; Purine-nucleoside phosphorylasedeficiency; Pyruvate carboxylase deficiency; Pyruvate dehydrogenaseE1-alpha deficiency; Pyruvate kinase deficiency of red cells; Rainesyndrome; Rasopathy; Recessive dystrophic epidermolysis bullosa; Naildisorder, nonsyndromic congenital, 8; Reifenstein syndrome; Renaladysplasia; Renal carnitine transport defect; Renal coloboma syndrome;Renal dysplasia; Renal dysplasia, retinal pigmentary dystrophy,cerebellar ataxia and skeletal dysplasia; Renal tubular acidosis,distal, autosomal recessive, with late-onset sensorineural hearing loss,or with hemolytic anemia; Renal tubular acidosis, proximal, with ocularabnormalities and mental retardation; Retinal cone dystrophy 3B;Retinitis pigmentosa; Retinitis pigmentosa 10, 11, 12, 14, 15, 17, and19; Retinitis pigmentosa 2, 20, 25, 35, 36, 38, 39, 4, 40, 43, 45, 48,66, 7, 70, 72; Retinoblastoma; Rett disorder; Rhabdoid tumorpredisposition syndrome 2; Rhegmatogenous retinal detachment, autosomaldominant; Rhizomelic chondrodysplasia punctata type 2 and type 3;Roberts-SC phocomelia syndrome; Robinow Sorauf syndrome; Robinowsyndrome, autosomal recessive, autosomal recessive, withbrachy-syn-polydactyly; Rothmund-Thomson syndrome; Rapadilino syndrome;RRM2B-related mitochondrial disease; Rubinstein-Taybi syndrome; Salladisease; Sandhoff disease, adult and infantil types; Sarcoidosis,early-onset; Blau syndrome; Schindler disease, type 1; Schizencephaly;Schizophrenia 15; Schneckenbecken dysplasia; Schwannomatosis 2; SchwartzJampel syndrome type 1; Sclerocornea, autosomal recessive;Sclerosteosis; Secondary hypothyroidism; Segawa syndrome, autosomalrecessive; Senior-Loken syndrome 4 and 5; Sensory ataxic neuropathy,dysarthria, and ophthalmoparesis; Sepiapterin reductase deficiency;SeSAME syndrome; Severe combined immunodeficiency due to ADA deficiency,with microcephaly, growth retardation, and sensitivity to ionizingradiation, atypical, autosomal recessive, T cell-negative, Bcell-positive, NK cell-negative of NK-positive; Severe congenitalneutropenia; Severe congenital neutropenia 3, autosomal recessive ordominant; Severe congenital neutropenia and 6, autosomal recessive;Severe myoclonic epilepsy in infancy; Generalized epilepsy with febrileseizures plus, types 1 and 2; Severe X-linked myotubular myopathy; ShortQT syndrome 3; Short stature with nonspecific skeletal abnormalities;Short stature, auditory canal atresia, mandibular hypoplasia, skeletalabnormalities; Short stature, onychodysplasia, facial dysmorphism, andhypotrichosis; Primordial dwarfism; Short-rib thoracic dysplasia 11 or 3with or without polydactyly; Sialidosis type I and II; Silver spasticparaplegia syndrome; Slowed nerve conduction velocity, autosomaldominant; Smith-Lemli-Opitz syndrome; Snyder Robinson syndrome;Somatotroph adenoma; Prolactinoma; familial, Pituitary adenomapredisposition; Sotos syndrome 1 or 2; Spastic ataxia 5, autosomalrecessive, Charlevoix-Saguenay type, 1,10, or 11, autosomal recessive;Amyotrophic lateral sclerosis type 5; Spastic paraplegia 15, 2, 3, 35,39, 4, autosomal dominant, 55, autosomal recessive, and 5A; Bile acidsynthesis defect, congenital, 3; Spermatogenic failure 11, 3, and 8;Spherocytosis types 4 and 5; Spheroid body myopathy; Spinal muscularatrophy, lower extremity predominant 2, autosomal dominant; Spinalmuscular atrophy, type II; Spinocerebellar ataxia 14, 21, 35, 40,and 6;Spinocerebellar ataxia autosomal recessive 1 and 16; Splenic hypoplasia;Spondylocarpotarsal synostosis syndrome; Spondylocheirodysplasia,Ehlers-Danlos syndrome-like, with immune dysregulation, Aggrecan type,with congenital joint dislocations, short limb-hand type, Sedaghatiantype, with cone-rod dystrophy, and Kozlowski type; Parastremmaticdwarfism; Stargardt disease 1; Cone-rod dystrophy 3; Stickler syndrometype 1; Kniest dysplasia; Stickler syndrome, types 1 (nonsyndromicocular) and 4; Sting-associated vasculopathy, infantile-onset;Stormorken syndrome; Sturge-Weber syndrome, Capillary malformations,congenital, 1; Succinyl-CoA acetoacetate transferase deficiency;Sucrase-isomaltase deficiency; Sudden infant death syndrome; Sulfiteoxidase deficiency, isolated; Supravalvar aortic stenosis; Surfactantmetabolism dysfunction, pulmonary, 2 and 3; Symphalangism, proximal, 1b;Syndactyly Cenani Lenz type; Syndactyly type 3; Syndromic X-linkedmental retardation 16; Talipes equinovarus; Tangier disease; TARPsyndrome; Tay-Sachs disease, B1 variant, Gm2-gangliosidosis (adult),Gm2-gangliosidosis (adult-onset); Temtamy syndrome; Tenorio Syndrome;Terminal osseous dysplasia; Testosterone 17-beta-dehydrogenasedeficiency; Tetraamelia, autosomal recessive; Tetralogy of Fallot;Hypoplastic left heart syndrome 2; Truncus arteriosus; Malformation ofthe heart and great vessels; Ventricular septal defect 1; Thiel-Behnkecorneal dystrophy; Thoracic aortic aneurysms and aortic dissections;Marfanoid habitus; Three M syndrome 2; Thrombocytopenia, plateletdysfunction, hemolysis, and imbalanced globin synthesis;Thrombocytopenia, X-linked; Thrombophilia, hereditary, due to protein Cdeficiency, autosomal dominant and recessive; Thyroid agenesis; Thyroidcancer, follicular; Thyroid hormone metabolism, abnormal; Thyroidhormone resistance, generalized, autosomal dominant; Thyrotoxic periodicparalysis and Thyrotoxic periodic paralysis 2; Thyrotropin-releasinghormone resistance, generalized; Timothy syndrome; TNFreceptor-associated periodic fever syndrome (TRAPS); Tooth agenesis,selective, 3 and 4; Torsades de pointes;Townes-Brocks-branchiootorenal-like syndrome; Transient bullousdermolysis of the newborn; Treacher collins syndrome 1; Trichomegalywith mental retardation, dwarfism and pigmentary degeneration of retina;Trichorhinophalangeal dysplasia type I; Trichorhinophalangeal syndrometype 3; Trimethylaminuria; Tuberous sclerosis syndrome;Lymphangiomyomatosis; Tuberous sclerosis 1 and 2; Tyrosinase-negativeoculocutaneous albinism; Tyrosinase-positive oculocutaneous albinism;Tyrosinemia type I; UDPglucose-4-epimerase deficiency; Ullrichcongenital muscular dystrophy; Ulna and fibula absence of with severelimb deficiency; Upshaw-Schulman syndrome; Urocanate hydratasedeficiency; Usher syndrome, types 1, 1B, 1D, 1G, 2A, 2C, and 2D;Retinitis pigmentosa 39; UV-sensitive syndrome; Van der Woude syndrome;Van Maldergem syndrome 2; Hennekam lymphangiectasia-lymphedema syndrome2; Variegate porphyria; Ventriculomegaly with cystic kidney disease;Verheij syndrome; Very long chain acyl-CoA dehydrogenase deficiency;Vesicoureteral reflux 8; Visceral heterotaxy 5, autosomal; Visceralmyopathy; Vitamin D-dependent rickets, types land 2; Vitelliformdystrophy; von Willebrand disease type 2M and type 3; Waardenburgsyndrome type 1, 4C, and 2E (with neurologic involvement);Klein-Waardenberg syndrome; Walker-Warburg congenital musculardystrophy; Warburg micro syndrome 2 and 4; Warts, hypogammaglobulinemia,infections, and myelokathexis; Weaver syndrome; Weill-Marchesanisyndrome 1 and 3; Weill-Marchesani-like syndrome;Weissenbacher-Zweymuller syndrome; Werdnig-Hoffmann disease;Charcot-Marie-Tooth disease; Werner syndrome; WFS1-Related Disorders;Wiedemann-Steiner syndrome; Wilson disease; Wolfram-like syndrome,autosomal dominant; Worth disease; Van Buchem disease type 2; Xerodermapigmentosum, complementation group b, group D, group E, and group G;X-linked agammaglobulinemia; X-linked hereditary motor and sensoryneuropathy; X-linked ichthyosis with steryl-sulfatase deficiency;X-linked periventricular heterotopia; Oto-palato-digital syndrome, typeI; X-linked severe combined immunodeficiency; Zimmermann-Laband syndromeand Zimmermann-Laband syndrome 2; and Zonular pulverulent cataract 3.

The target nucleotide sequence may comprise a target sequence (e.g., apoint mutation) associated with a disease, disorder, or condition. Thetarget sequence may comprise a T to C (or A to G) point mutationassociated with a disease, disorder, or condition, and wherein thedeamination of the mutant C base results in mismatch repair-mediatedcorrection to a sequence that is not associated with a disease,disorder, or condition. The target sequence may comprise a G to A (or Cto T) point mutation associated with a disease, disorder, or condition,and wherein the deamination of the mutant A base results in mismatchrepair-mediated correction to a sequence that is not associated with adisease, disorder, or condition. The target sequence may encode aprotein, and where the point mutation is in a codon and results in achange in the amino acid encoded by the mutant codon as compared to awild-type codon. The target sequence may also be at a splice site, andthe point mutation results in a change in the splicing of an mRNAtranscript as compared to a wild-type transcript. In addition, thetarget may be at a non-coding sequence of a gene, such as a promoter,and the point mutation results in increased or decreased expression ofthe gene.

Thus, in some aspects, the deamination of a mutant C results in a changeof the amino acid encoded by the mutant codon, which in some cases canresult in the expression of a wild-type amino acid. In other aspects,the deamination of a mutant A results in a change of the amino acidencoded by the mutant codon, which in some cases can result in theexpression of a wild-type amino acid.

The methods described herein involving contacting a cell with acomposition or rAAV particle can occur in vitro, ex vivo, or in vivo. Incertain embodiments, the step of contacting occurs in a subject. Incertain embodiments, the subject has been diagnosed with a disease,disorder, or condition.

In some embodiments, the methods disclosed herein involve contacting amammalian cell with a composition or rAAV particle. In particularembodiments, the methods involve contacting a retinal cell, corticalcell or cerebellar cell.

The split Cas9 protein or split prime editor delivered using the methodsdescribed herein preferably have comparable activity compared to theoriginal Cas9 protein or prime editor (i.e., unsplit protein deliveredto a cell or expressed in a cell as a whole). For example, the splitCas9 protein or split prime editor retains at least 50% (e.g., at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or 100%) of the activity of theoriginal Cas9 protein or prime editor. In some embodiments, the splitCas9 protein or split prime editor is more active (e.g., 2-fold, 5-fold,10-fold, 100-fold, 1000-fold, or more) than that of an original Cas9protein or prime editor.

The compositions described herein may be administered to a subject inneed thereof in a therapeutically effective amount to treat and/orprevent a disease or disorder the subject is suffering from. Any diseaseor disorder that maybe treated and/or prevented using CRISPR/Cas9-basedgenome-editing technology may be treated by the split Cas9 protein orthe split prime editor described herein. It is to be understood that, ifthe nucleotide sequences encoding the split Cas9 protein or the primeeditor does not further encode a gRNA, a separate nucleic acid vectorencoding the gRNA may be administered together with the compositionsdescribed herein.

Exemplary suitable diseases, disorders or conditions include, withoutlimitation the disease or disorder is selected from the group consistingof: cystic fibrosis, phenylketonuria, epidermolytic hyperkeratosis(EHK), chronic obstructive pulmonary disease (COPD), Charcot-Marie-Tootdisease type 4J, neuroblastoma (NB), von Willebrand disease (vWD),myotonia congenital, hereditary renal amyloidosis, dilatedcardiomyopathy, hereditary lymphedema, familial Alzheimer's disease,prion disease, chronic infantile neurologic cutaneous articular syndrome(CINCA), congenital deafness, Niemann-Pick disease type C (NPC) disease,and desmin-related myopathy (DRM). In particular embodiments, thedisease or condition is Niemann-Pick disease type C (NPC) disease.

In some embodiments, the disease, disorder or condition is associatedwith a point mutation in an NPC gene, a DNMT1 gene, a PCSK9 gene, or aTMC1 gene. In certain embodiments, the point mutation is a T3182Cmutation in NPC, which results in an I1061T amino acid substitution.

In certain embodiments, the point mutation is an A545G mutation in TMC1,which results in a Y182C amino acid substitution. TMC1 encodes a proteinthat forms mechanosensitive ion channels in sensory hair cells of theinner ear and is required for normal auditory function. The Y182C aminoacid substitution is associated with congenital deafness.

In some embodiments, the disease, disorder or condition is associatedwith a point mutation that generates a stop codon, for example, apremature stop codon within the coding region of a gene.

Additional exemplary diseases, disorders and conditions include cysticfibrosis (see, e.g., Schwank et al., Functional repair of CFTR byCRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosispatients. Cell stem cell. 2013; 13: 653-658; and Wu et. al., Correctionof a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell.2013; 13: 659-662, neither of which uses a deaminase fusion protein tocorrect the genetic defect); phenylketonuria—e.g., phenylalanine toserine mutation at position 835 (mouse) or 240 (human) or a homologousresidue in phenylalanine hydroxylase gene (T>C mutation)—see, e.g.,McDonald et al., Genomics. 1997; 39:402-405; Bernard-Soulier syndrome(BSS)—e.g., phenylalanine to serine mutation at position 55 or ahomologous residue, or cysteine to arginine at residue 24 or ahomologous residue in the platelet membrane glycoprotein IX (T>Cmutation)—see, e.g., Noris et al., British Journal of Haematology. 1997;97: 312-320, and Ali et al., Hematol. 2014; 93: 381-384; epidermolytichyperkeratosis (EHK)—e.g., leucine to proline mutation at position 160or 161 (if counting the initiator methionine) or a homologous residue inkeratin 1 (T>C mutation)—see, e.g., Chipev et al., Cell. 1992; 70:821-828, see also accession number P04264 in the UNIPROT database atwww[dot]uniprot[dot]org; chronic obstructive pulmonary disease(COPD)—e.g., leucine to proline mutation at position 54 or 55 (ifcounting the initiator methionine) or a homologous residue in theprocessed form of α₁-antitrypsin or residue 78 in the unprocessed formor a homologous residue (T>C mutation)—see, e.g., Poller et al.,Genomics. 1993; 17: 740-743, see also accession number P01011 in theUNIPROT database; Charcot-Marie-Toot disease type 4J—e.g., isoleucine tothreonine mutation at position 41 or a homologous residue in FIG. 4 (T>Cmutation)—see, e.g., Lenk et al., PLoS Genetics. 2011; 7: e1002104;neuroblastoma (NB)—e.g., leucine to proline mutation at position 197 ora homologous residue in Caspase-9 (T>C mutation)—see, e.g., Kundu etal., 3 Biotech. 2013, 3:225-234; von Willebrand disease (vWD)—e.g.,cysteine to arginine mutation at position 509 or a homologous residue inthe processed form of von Willebrand factor, or at position 1272 or ahomologous residue in the unprocessed form of von Willebrand factor (T>Cmutation)—see, e.g., Lavergne et al., Br. J. Haematol. 1992, see alsoaccession number P04275 in the UNIPROT database; 82: 66-72; myotoniacongenital—e.g., cysteine to arginine mutation at position 277 or ahomologous residue in the muscle chloride channel gene CLCN1 (T>Cmutation)—see, e.g., Weinberger et al., The J. of Physiology. 2012; 590:3449-3464; hereditary renal amyloidosis—e.g., stop codon to argininemutation at position 78 or a homologous residue in the processed form ofapolipoprotein All or at position 101 or a homologous residue in theunprocessed form (T>C mutation)—see, e.g., Yazaki et al., Kidney Int.2003; 64: 11-16; dilated cardiomyopathy (DCM)—e.g., tryptophan toArginine mutation at position 148 or a homologous residue in the FOXD4gene (T>C mutation), see, e.g., Minoretti et. al., Int. J. of Mol. Med.2007; 19: 369-372; hereditary lymphedema—e.g., histidine to argininemutation at position 1035 or a homologous residue in VEGFR3 tyrosinekinase (A>G mutation), see, e.g., Irrthum et al., Am. J. Hum. Genet.2000; 67: 295-301; familial Alzheimer's disease—e.g., isoleucine tovaline mutation at position 143 or a homologous residue in presenilin1(A>G mutation), see, e.g., Gallo et. al., J. Alzheimer's disease. 2011;25: 425-431; Prion disease—e.g., methionine to valine mutation atposition 129 or a homologous residue in prion protein (A>Gmutation)—see, e.g., Lewis et. al., J. of General Virology. 2006; 87:2443-2449; chronic infantile neurologic cutaneous articular syndrome(CINCA)—e.g., Tyrosine to Cysteine mutation at position 570 or ahomologous residue in cryopyrin (A>G mutation)—see, e.g., Fujisawa et.al. Blood. 2007; 109: 2903-2911; and desmin-related myopathy (DRM)—e.g.,arginine to glycine mutation at position 120 or a homologous residue inαβ crystallin (A>G mutation)—see, e.g., Kumar et al., J. Biol. Chem.1999; 274: 24137-24141. The entire contents of all references anddatabase entries is incorporated herein by reference.

Trinucleotide Repeat Expansion Disease

Trinucleotide repeat expansion is associated with a number of humandiseases, including Huntington's Disease, Fragile X syndrome, andFriedreich's ataxia. The most common trinucleotide repeat contains CAGtriplets, though GAA triplets (Friedreich's ataxia) and CGG triplets(Fragile X syndrome) also occur. Inheriting a predisposition toexpansion, or acquiring an already expanded parental allele, increasesthe likelihood of acquiring the disease. Pathogenic expansions oftrinucleotide repeats could hypothetically be corrected using primeediting.

A region upstream of the repeat region can be nicked by an RNA-guidednuclease, then used to prime synthesis of a new DNA strand that containsa healthy number of repeats (which depends on the particular gene anddisease), in accordance with the general mechanism outlined in FIG. 1Gor FIG. 22 . After the repeat sequence, a short stretch of homology isadded that matches the identity of the sequence adjacent to the otherend of the repeat (bold strand). Invasion of the newly synthesizedstrand by the TPRT system, and subsequent replacement of the endogenousDNA with the newly synthesized flap, leads to a contracted repeatallele. The term “contracted” refers to a shortening of the length ofthe nucleotide repeat region, thereby resulting in repairing thetrinucleotide repeat region.

The prime editing system or prime editing (PE) system described hereinmay be used to contract trinucleotide repeat mutations (or “tripletexpansion diseases”) to treating conditions such as Huntington's diseaseand other trinucleotide repeat disorders. Trinucleotide repeat expansiondisorders are complex, progressive disorders that involve developmentalneurobiology and often affect cognition as well as sensori-motorfunctions. The disorders show genetic anticipation (i.e. increasedseverity with each generation). The DNA expansions or contractionsusually happen meiotically (i.e. during the time of gametogenesis, orearly in embryonic development), and often have sex-bias meaning thatsome genes expand only when inherited through the female, others onlythrough the male. In humans, trinucleotide repeat expansion disorderscan cause gene silencing at either the transcriptional or translationallevel, which essentially knocks out gene function. Alternatively,trinucleotide repeat expansion disorders can cause altered proteinsgenerated with large repetitive amino acid sequences that eitherabrogate or change protein function, often in a dominant-negative manner(e.g. poly-glutamine diseases).

Without wishing to be bound by theory, triplet expansion is caused byslippage during DNA replication or during DNA repair synthesis. Becausethe tandem repeats have identical sequence to one another, base pairingbetween two DNA strands can take place at multiple points along thesequence. This may lead to the formation of “loop out” structures duringDNA replication or DNA repair synthesis. This may lead to repeatedcopying of the repeated sequence, expanding the number of repeats.Additional mechanisms involving hybrid RNA:DNA intermediates have beenproposed. Prime editing may be used to reduce or eliminate these tripletexpansion regions by deletion one or more or the offending repeat codontriplets. In an embodiment of this use, FIG. 23 , provides a schematicof a PEgRNA design for contracting or reducing trinucleotide repeatsequences with prime editing.

Prime editing may be implemented to contract triplet expansion regionsby nicking a region upstream of the triplet repeat region with the primeeditor comprising a PEgRNA appropriated targeted to the cut site. Theprime editor then synthesizes a new DNA strand (ssDNA flap) based on thePEgRNA as a template (i.e., the edit template thereof) that codes for ahealthy number of triplet repeats (which depends on the particular geneand disease). The newly synthesized ssDNA strand comprising the healthytriplet repeat sequence also is synthesized to include a short stretchof homology (i.e., the homology arm) that matches the sequence adjacentto the other end of the repeat (bold strand). Invasion of the newlysynthesized strand, and subsequent replacement of the endogenous DNAwith the newly synthesized ssDNA flap, leads to a contracted repeatallele.

Depending on the particular trinucleotide expansion disorder, thedefect-inducing triplet expansions may occur in “trinucleotide repeatexpansion proteins.” Trinucleotide repeat expansion proteins are adiverse set of proteins associated with susceptibility for developing atrinucleotide repeat expansion disorder, the presence of a trinucleotiderepeat expansion disorder, the severity of a trinucleotide repeatexpansion disorder or any combination thereof. Trinucleotide repeatexpansion disorders are divided into two categories determined by thetype of repeat. The most common repeat is the triplet CAG, which, whenpresent in the coding region of a gene, codes for the amino acidglutamine (Q). Therefore, these disorders are referred to as thepolyglutamine (polyQ) disorders and comprise the following diseases:Huntington Disease (HD); Spinobulbar Muscular Atrophy (SBMA);Spinocerebellar Ataxias (SCA types 1, 2, 3, 6, 7, and 17); andDentatorubro-Pallidoluysian Atrophy (DRPLA). The remaining trinucleotiderepeat expansion disorders either do not involve the CAG triplet or theCAG triplet is not in the coding region of the gene and are, therefore,referred to as the non-polyglutamine disorders. The non-polyglutaminedisorders comprise Fragile X Syndrome (FRAXA); Fragile XE MentalRetardation (FRAXE); Friedreich Ataxia (FRDA); Myotonic Dystrophy (DM);and Spinocerebellar Ataxias (SCA types 8, and 12).

The proteins associated with trinucleotide repeat expansion disorderscan be selected based on an experimental association of the proteinassociated with a trinucleotide repeat expansion disorder to atrinucleotide repeat expansion disorder. For example, the productionrate or circulating concentration of a protein associated with atrinucleotide repeat expansion disorder may be elevated or depressed ina population having a trinucleotide repeat expansion disorder relativeto a population lacking the trinucleotide repeat expansion disorder.Differences in protein levels may be assessed using proteomic techniquesincluding but not limited to Western blot, immunohistochemical staining,enzyme linked immunosorbent assay (ELISA), and mass spectrometry.Alternatively, the proteins associated with trinucleotide repeatexpansion disorders may be identified by obtaining gene expressionprofiles of the genes encoding the proteins using genomic techniquesincluding but not limited to DNA microarray analysis, serial analysis ofgene expression (SAGE), and quantitative real-time polymerase chainreaction (Q-PCR).

Non-limiting examples of proteins associated with trinucleotide repeatexpansion disorders which can be corrected by prime editing include AR(androgen receptor), FMR1 (fragile X mental retardation 1), HTT(huntingtin), DMPK (dystrophia myotonica-protein kinase), FXN(frataxin), ATXN2 (ataxin 2), ATN1 (atrophin 1), FEN1 (flapstructure-specific endonuclease 1), TNRC6A (trinucleotide repeatcontaining 6A), PABPN1 (poly(A) binding protein, nuclear 1), JPH3(junctophilin 3), MED15 (mediator complex subunit 15), ATXN1 (ataxin 1),ATXN3 (ataxin 3), TBP (TATA box binding protein), CACNA1A (calciumchannel, voltage-dependent, P/Q type, alpha 1A subunit), ATXN80S (ATXN8opposite strand (non-protein coding)), PPP2R2B (protein phosphatase 2,regulatory subunit B, beta), ATXN7 (ataxin 7), TNRC6B (trinucleotiderepeat containing 6B), TNRC6C (trinucleotide repeat containing 6C),CELF3 (CUGBP, Elav-like family member 3), MAB21L1 (mab-21-like 1 (C.elegans)), MSH2 (mutS homolog 2, colon cancer, nonpolyposis type 1 (E.coli)), TMEM185A (transmembrane protein 185A), SIX5 (SIX homeobox 5),CNPY3 (canopy 3 homolog (zebrafish)), FRAXE (fragile site, folic acidtype, rare, fra(X)(q28) E), GNB2 (guanine nucleotide binding protein (Gprotein), beta polypeptide 2), RPL14 (ribosomal protein L14), ATXN8(ataxin 8), INSR (insulin receptor), TTR (transthyretin), EP400 (E1Abinding protein p400), GIGYF2 (GRB10 interacting GYF protein 2), OGG1(8-oxoguanine DNA glycosylase), STC1 (stanniocalcin 1), CNDP1 (carnosinedipeptidase 1 (metallopeptidase M20 family)), C10orf2 (chromosome 10open reading frame 2), MAML3 mastermind-like 3 (Drosophila), DKC1(dyskeratosis congenita 1, dyskerin), PAXIP1 (PAX interacting (withtranscription-activation domain) protein 1), CASK(calcium/calmodulin-dependent serine protein kinase (MAGUK family)),MAPT (microtubule-associated protein tau), SP1 (Sp1 transcriptionfactor), POLG (polymerase (DNA directed), gamma), AFF2 (AF4/FMR2 family,member 2), THBS1 (thrombospondin 1), TP53 (tumor protein p53), ESR1(estrogen receptor 1), CGGBP1 (CGG triplet repeat binding protein 1),ABT1 (activator of basal transcription 1), KLK3 (kallikrein-relatedpeptidase 3), PRNP (prion protein), JUN (jun oncogene), KCNN3 (potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 3), BAX (BCL2-associated X protein), FRAXA (fragile site, folicacid type, rare, fra(Xxq27.3) A (macroorchidism, mental retardation)),KBTBD10 (kelch repeat and BTB (POZ) domain containing 10), MBNL1(muscleblind-like (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, E.coli) (S. cerevisiae)), NCOA3 (nuclear receptor coactivator 3), ERDA1(expanded repeat domain, CAG/CTG 1), TSC1 (tuberous sclerosis 1), COMP(cartilage oligomeric matrix protein), GCLC (glutamate-cysteine ligase,catalytic subunit), RRAD (Ras-related associated with diabetes), MSH3(mutS homolog 3 (E. coli)), DRD2 (dopamine receptor D2), CD44 (CD44molecule (Indian blood group)), CTCF (CCCTC-binding factor (zinc fingerprotein)), CCND1 (cyclin D1), CLSPN (claspin homolog (Xenopus laevis)),MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase,receptor type, U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase),TRIM22 (tripartite motif-containing 22), WT1 (Wilms tumor 1), AHR (arylhydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurineS-methyltransferase), NDP (Norrie disease (pseudoglioma)), ARX(aristaless related homeobox), MUS81 (MUS81 endonuclease homolog (S.cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (earlygrowth response 1), UNG (uracil-DNA glycosylase), NUMBL (numb homolog(Drosophila)-like), FABP2 (fatty acid binding protein 2, intestinal),EN2 (engrailed homeobox 2), CRYGC (crystallin, gamma C), SRP14 (signalrecognition particle 14 kDa (homologous Alu RNA binding protein)), CRYGB(crystallin, gamma B), PDCD1 (programmed cell death 1), HOXA1 (homeoboxA1), ATXN2L (ataxin 2-like), PMS2 (PMS2 postmeiotic segregationincreased 2 (S. cerevisiae)), GLA (galactosidase, alpha), CBL (Cas-Br-M(murine) ecotropic retroviral transforming sequence), FTH1 (ferritin,heavy polypeptide 1), IL12RB2 (interleukin 12 receptor, beta 2), OTX2(orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNAdirected), gamma 2, accessory subunit), DLX2 (distal-less homeobox 2),SIRPA (signal-regulatory protein alpha), OTX1 (orthodenticle homeobox1), AHRR (aryl-hydrocarbon receptor repressor), MANF (mesencephalicastrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein158 (gene/pseudogene)), and ENSG00000078687.

In a particular aspect, the instant disclosure provides TPRT-basedmethods for the treatment of a subject diagnosed with an expansionrepeat disorder (also known as a repeat expansion disorder or atrinucleotide repeat disorder). Expansion repeat disorders occur whenmicrosatellite repeats expand beyond a threshold length. Currently, atleast 30 genetic diseases are believed to be caused by repeatexpansions. Scientific understanding of this diverse group of disorderscame to lights in the early 1990's with the discovery that trinucleotiderepeats underlie several major inherited conditions, including FragileX, Spinal and Bulbar Muscular Atrophy, Myotonic Dystrophy, andHuntington's disease (Nelson et al, “The unstable repeats—three evolvingfaces of neurological disease,” Neuron, Mar. 6, 2013, Vol. 77; 825-843,which is incorporated herein by reference), as well as Haw RiverSyndrome, Jacobsen Syndrome, Dentatorubral-pallidoluysian atrophy(DRPLA), Machado-Joseph disease, Synpolydactyly (SPD II), Hand-footgenital syndrome (HFGS), Cleidocranial dysplasia (CCD),Holoprosencephaly disorder (HPE), Congenital central hypventilationsyndrome (CCHS), ARX-nonsyndromic X-linked mental retardation (XLMR),and Oculopharyngeal muscular dystrophy (OPMD) (see. Microsatelliterepeat instability was found to be a hallmark of these conditions, aswas anticipation—the phenomenon in which repeat expansion can occur witheach successive generation, which leads to a more severe phenotype andearlier age of onset in the offspring. Repeat expansions are believed tocause diseases via several different mechanisms. Namely, expansions mayinterfere with cellular functioning at the level of the gene, the mRNAtranscript, and/or the encoded protein. In some conditions, mutationsact via a loss-of-function mechanism by silencing repeat-containinggenes. In others, disease results from gain-of-function mechanisms,whereby either the mRNA transcript or protein takes on new, aberrantfunctions.

In one embodiment, a method of treating a trinucleotide repeat disorderis depicted in FIG. 23 . In general, the approach involves using TPRTgenome editing (i.e., prime editing) in combination with an extendedgRNA that comprises a region that encodes a desired and healthyreplacement trinucleotide repeat sequence that is intended to replacethe endogenous diseased trinucleotide repeat sequence through themechanism of the prime editing process. A schematic of an exemplary gRNAdesign for contracting trinucleotide repeat sequences and trinucleotiderepeat contraction with TPRT genome editing (i.e., prime editing) isshown in FIG. 23 .

Prion Disease

Prime editing can also be used to prevent or halt the progression ofprion disease through the installation of one or more protectivemutations into prion proteins (PRNP) which become misfolded during thecourse of disease. Prion diseases or transmissible spongiformencephalopathies (TSEs) are a family of rare progressiveneurodegenerative disorders that affect both humans and animals. Theyare distinguished by long incubation periods, characteristic spongiformchanges associated with neuronal loss, and a failure to induceinflammatory response.

In humans, prion disease includes Creutzfeldt-Jakob Disease (CJD),Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-ScheinkerSyndrome, Fatal Familial Insomnia, and Kuru. In animals, prion diseaseincludes Bovine Spongiform Encephalopathy (BSE or “mad cow disease”),Chronic Wasting Disease (CWD), Scrapie, Transmissible MinkEncephalopathy, Feline Spongiform Encephalopathy, and UngulateSpongiform Encephalopathy. Prime editing may be used to installprotective point mutations into a prion protein in order to prevent orhalt the progression of any one of these prion diseases.

Classic CJD is a human prion disease. It is a neurodegenerative disorderwith characteristic clinical and diagnostic features. This disease israpidly progressive and always fatal. Infection with this disease leadsto death usually within 1 year of onset of illness. CJD is a rapidlyprogressive, invariably fatal neurodegenerative disorder believed to becaused by an abnormal isoform of a cellular glycoprotein known as theprion protein. CJD occurs worldwide and the estimated annual incidencein many countries, including the United States, has been reported to beabout one case per million population. The vast majority of CJD patientsusually die within 1 year of illness onset. CJD is classified as atransmissible spongiform encephalopathy (TSE) along with other priondiseases that occur in humans and animals. In about 85% of patients, CJDoccurs as a sporadic disease with no recognizable pattern oftransmission. A smaller proportion of patients (5 to 15%) develop CJDbecause of inherited mutations of the prion protein gene. Theseinherited forms include Gerstmann-Straussler-Scheinker syndrome andfatal familial insomnia. No treatment is currently known for CJD.

Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease that wasfirst described in 1996 in the United Kingdom. There is now strongscientific evidence that the agent responsible for the outbreak of priondisease in cows, bovine spongiform encephalopathy (BSE or ‘mad cow’disease), is the same agent responsible for the outbreak of vCJD inhumans. Variant CJD (vCJD) is not the same disease as classic CJD (oftensimply called CJD). It has different clinical and pathologiccharacteristics from classic CJD. Each disease also has a particulargenetic profile of the prion protein gene. Both disorders are invariablyfatal brain diseases with unusually long incubation periods measured inyears and are caused by an unconventional transmissible agent called aprion. No treatment is currently known for vCJD.

BSE (bovine spongiform encephalopathy or “mad cow disease”) is aprogressive neurological disorder of cattle that results from infectionby an unusual transmissible agent called a prion. The nature of thetransmissible agent is not well understood. Currently, the most acceptedtheory is that the agent is a modified form of a normal protein known asprion protein. For reasons that are not yet understood, the normal prionprotein changes into a pathogenic (harmful) form that then damages thecentral nervous system of cattle. There is increasing evidence thatthere are different strains of BSE: the typical or classic BSE strainresponsible for the outbreak in the United Kingdom and two atypicalstrains (H and L strains). No treatment is currently known for BSE.

Chronic wasting disease (CWD) is a prion disease that affects deer, elk,reindeer, sika deer and moose. It has been found in some areas of NorthAmerica, including Canada and the United States, Norway and South Korea.It may take over a year before an infected animal develops symptoms,which can include drastic weight loss (wasting), stumbling, listlessnessand other neurologic symptoms. CWD can affect animals of all ages andsome infected animals may die without ever developing the disease. CWDis fatal to animals and there are no treatments or vaccines.

The causative agents of TSEs are believed to be prions. The term“prions” refers to abnormal, pathogenic agents that are transmissibleand are able to induce abnormal folding of specific normal cellularproteins called prion proteins that are found most abundantly in thebrain. The functions of these normal prion proteins are still notcompletely understood. The abnormal folding of the prion proteins leadsto brain damage and the characteristic signs and symptoms of thedisease. Prion diseases are usually rapidly progressive and alwaysfatal.

As used herein, the term “prion” shall mean an infectious particle knownto cause diseases (spongiform encephalopathies) in humans and animals.The term “prion” is a contraction of the words “protein” and “infection”and the particles are comprised largely if not exclusively of PRNP^(Sc)molecules encoded by a PRNP gene which expresses PRNP^(C) which changesconformation to become PRNP^(Sc). Prions are distinct from bacteria,viruses and viroids. Known prions include those which infect animals tocause scrapie, a transmissible, degenerative disease of the nervoussystem of sheep and goats as well as bovine spongiform encephalopathies(BSE) or mad cow disease and feline spongiform encephalopathies of cats.Four prion diseases, as discussed above, known to affect humans are (1)kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI). As used herein prion includes all forms of prionscausing all or any of these diseases or others in any animals used—andin particular in humans and in domesticated farm animals.

In general, and without wishing to be bound by theory, prior diseasesare caused by misfolding of prion proteins. Such diseases-often calleddeposition diseases—the misfolding of the prion proteins can beaccounted for as follows. If A is the normally synthesized gene productthat carries out an intended physiologic role in a monomeric oroligomeric state, A* is a conformationally activated form of A that iscompetent to undergo a dramatic conformational change, B is theconformationally altered state that prefers multimeric assemblies (i.e.,the misfolded form which forms depositions) and B_(n) is the multimericmaterial that is pathogenic and relatively difficult to recycle. For theprion diseases, PRNP^(C) and PRNP^(Sc) correspond to states A and B_(n)where A is largely helical and monomeric and B_(n) is β-rich andmultimeric.

It is known that certain mutations in prion proteins can be associatedwith increased risk of prior disease. Conversely, certain mutations inprion proteins can be protective in nature. See Bagynszky et al.,“Characterization of mutations in PRNP (prion) gene and their possibleroles in neurodegenerative diseases,” Neuropsychiatr Dis Treat., 2018;14: 2067-2085, the contents of which are incorporated herein byreference.

PRNP (NCBI RefSeq No. NP_000302.1 (SEQ ID NO: 291))—the human prionprotein—is encoded by a 16 kb long gene, located on chromosome 20(4686151-4701588). It contains two exons, and the exon 2 carries theopen reading frame which encodes the 253 amino acid (AA) long PrPprotein. Exon 1 is a noncoding exon, which may serve as transcriptionalinitiation site. The post-translational modifications result in theremoval of the first 22 AA N-terminal fragment (NTF) and the last 23 AAC-terminal fragment (CTF). The NTF is cleaved after PrP transport to theendoplasmic reticulum (ER), while the CTF (glycosylphosphatidylinositol[GPI] signal peptide [GPI-SP]) is cleaved by the GPI anchor. GPI anchorcould be involved in PrP protein transport. It may also play a role ofattachment of prion protein into the outer surface of cell membrane.Normal PrP is composed of a long N-terminal loop (which contains theoctapeptide repeat region), two short p sheets, three a helices, and aC-terminal region (which contains the GPI anchor). Cleavage of PrPresults in a 208 AA long glycoprotein, anchored in the cell membrane.

The 253 amino acid sequence of PRNP (NP_000302.1) is as follows:

(SEQ ID NO: 291) MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFL IVG.

The 253 amino acid sequence of PRNP (NP_000302.1) is encoded by thefollowing nucleotide sequence (NCBI Ref. Seq No. NM_000311.5, “Homosapiens prion protein (PRNP), transcript variant 1, mRNA), is asfollows:

(SEQ ID NO: 292) GCGAACCTTGGCTGCTGGATGCTGGTTCTCTTTGTGGCCACATGGAGTGACCTGGGCCTCTGCAAGAAGCGCCCGAAGCCTGGAGGATGGAACACTGGGGGCAGCCGATACCCGGGGCAGGGCAGCCCTGGAGGCAACCGCTACCCACCTCAGGGCGGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCCCCATGGTGGTGGCTGGGGACAGCCTCATGGTGGTGGCTGGGGTCAAGGAGGTGGCACCCACAGTCAGTGGAACAAGCCGAGTAAGCCAAAAACCAACATGAAGCACATGGCTGGTGCTGCAGCAGCTGGGGCAGTGGTGGGGGGCCTTGGCGGCTACATGCTGGGAAGTGCCATGAGCAGGCCCATCATACATTTCGGCAGTGACTATGAGGACCGTTACTATCGTGAAAACATGCACCGTTACCCCAACCAAGTGTACTACAGGCCCATGGATGAGTACAGCAACCAGAACAACTTTGTGCACGACTGCGTCAATATCACAATCAAGCAGCACACGGTCACCACAACCACCAAGGGGGAGAACTTCACCGAGACCGACGTTAAGATGATGGAGCGCGTGGTTGAGCAGATGTGTATCACCCAGTACGAGAGGGAATCTCAGGCCTATTACCAGAGAGGATCGAGCATGGTCCTCTTCTCCTCTCCACCTGTGATCCTCCTGATCTCTTTCCTCATCTTCCTGATAGTGGGATGAGGAAGGTCTTCCTGTTTTCACCATCTTTCTAATCTTTTTCCAGCTTGAGGGAGGCGGTATCCACCTGCAGCCCTTTTAGTGGTGGTGTCTCACTCTTTCTTCTCTCTTTGTCCCGGATAGGCTAATCAATACCCTTGGCACTGATGGGCACTGGAAAACATAGAGTAGACCTGAGATGCTGGTCAAGCCCCCTTTGATTGAGTTCATCATGAGCCGTTGCTAATGCCAGGCCAGTAAAAGTATAACAGCAAATAACCATTGGTTAATCTGGACTTATTTTTGGACTTAGTGCAACAGGTTGAGGCTAAAACAAATCTCAGAACAGTCTGAAATACCTTTGCCTGGATACCTCTGGCTCCTTCAGCAGCTAGAGCTCAGTATACTAATGCCCTATCTTAGTAGAGATTTCATAGCTATTTAGAGATATTTTCCATTTTAAGAAAACCCGACAACATTTCTGCCAGGTTTGTTAGGAGGCCACATGATACTTATTCAAAAAAATCCTAGAGATTCTTAGCTCTTGGGATGCAGGCTCAGCCCGCTGGAGCATGAGCTCTGTGTGTACCGAGAACTGGGGTGATGTTTTACTTTTCACAGTATGGGCTACACAGCAGCTGTTCAACAAGAGTAAATATTGTCACAACACTGAACCTCTGGCTAGAGGACATATTCACAGTGAACATAACTGTAACATATATGAAAGGCTTCTGGGACTTGAAATCAAATGTTTGGGAATGGTGCCCTTGGAGGCAACCTCCCATTTTAGATGTTTAAAGGACCCTATATGTGGCATTCCTTTCTTTAAACTATAGGTAATTAAGGCAGCTGAAAAGTAAATTGCCTTCTAGACACTGAAGGCAAATCTCCTTTGTCCATTTACCTGGAAACCAGAATGATTTTGACATACAGGAGAGCTGCAGTTGTGAAAGCACCATCATCATAGAGGATGATGTAATTAAAAAATGGTCAGTGTGCAAAGAAAAGAACTGCTTGCATTTCTTTATTTCTGTCTCATAATTGTCAAAAACCAGAATTAGGTCAAGTTCATAGTTTCTGTAATTGGCTTTTGAATCAAAGAATAGGGAGACAATCTAAAAAATATCTTAGGTTGGAGATGACAGAAATATGATTGATTTGAAGTGGAAAAAGAAATTCTGTTAATGTTAATTAAAGTAAAATTATTCCCTGAATTGTTTGATATTGTCACCTAGCAGATATGTATTACTTTTCTGCAATGTTATTATTGGCTTGCACTTTGTGAGTATTCTATGTAAAAATATATATGTATATAAAATATATATTGCATAGGACAGACTTAGGAGTTTTGTTTAGAGCAGTTAACATCTGAAGTGTCTAATGCATTAACTTTTGTAAGGTACTGAATACTTAATATGTGGGAAACCCTTTTGCGTGGTCCTTAGGCTTACAATGTGCACTGAATCGTTTCATGTAAGAATCCAAAGTGGACACCATTAACAGGTCTTTGAAATATGCATGTACTTTATATTTTCTATATTTGTAACTTTGCATGTTCTTGTTTTGTTATATAAAAAAATTGTAAATGTTTAATATCTGACTGAAATTAAACGAG CGAAGATGAGCACCA

Mutation sites relative to PRNP (NP_000302.1) which are linked to CJDand FFI are reported are as follows. These mutations can be removed orinstalled using the prime editors disclosed herein.

AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO CJD PRIONDISEASE (SEE TABLE 1 OF BAGYNSZKY ET AL., 2018) (RELATIVE TO MUTATIONSEQ ID NO: 291 OF PRNP NP_000302.1) D178NMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHNCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3940)T188K MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHKVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3941)E196K MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGKNFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3942)E196A MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGANFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 296)E200K MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTKTDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3943)E200G MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTGTDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 298)V203I MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDIKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3944)R208H MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMEHVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3945)V210I MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVIEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3946)E211Q MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVQQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3947)M232R MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSRVLFSSPPV (SEQ ID NO: 3948)

Mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) which arelinked to GSS are reported, as follows:

AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO GSS PRIONDISEASE (SEE TABLE 2 OF BAGYNSZKY ET AL., 2018) (RELATIVE TO MUTATIONSEQ ID NO: 291 OF PRNP NP_000302.1) P102LMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKLSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3949)P105L MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKLKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3950)A117V MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAVAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3951)G131V MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLVSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3952)V176G MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFGHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3953)H187R MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQRTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3954)MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 4084)F198S MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENSTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3955)D202N MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETNVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3956)Q212P MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEPMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3957)Q217R MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITRYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3958)M232T MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSTVLFSSPPV (SEQ ID NO: 3959)

Mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) which arelinked to a possible protective nature against prion disease, asfollows:

AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO A PROTECTIVENATURE AGAINST PRION DISEASE (SEE TABLE 4 OF BAGYNSZKY ET MUTATIONAL., 2018) (RELATIVE TO SEQ ID NO: 291 OF PRNP NP_000302.1) G127SMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGSYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3960)G127V MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGVYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3961)M129V MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYVLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3962)D167G MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMGEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3963)D167N MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMNEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3964)N171S MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSSQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3965)E219K MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYKRESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 3966)P238S MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSSPV (SEQ ID NO: 3967)

Thus, in various embodiments, prime editing may be used to remove amutation in PRNP that is linked to prion disease or install a mutationin PRNP that is considered to be protective against prion disease. Forexample, prime editing may be use to remove or restore a D178N, V180I,T188K, E196K, E196A, E200K, E200G, V203I, R208H, V210I, E211Q, I215V, orM232R mutation in the PRNP protein (relative to PRNP of NP_000302.1)(SEQ ID NO: 291). In other embodiments, prime editing may be use toremove or restore a P102L, P105L, A117V, G131V, V176G, H187R, F198S,D202N, Q212P, Q217R, or M232T mutation in the PRNP protein (relative toPRNP of NP_000302.1) (SEQ ID NO: 291). By removing or correcting for thepresence of such mutations in PRNP using prime editing, the risk ofprion disease may be reduced or eliminated.

In other embodiments, prime editing may be used to install a protectivemutation in PRNP that is linked to a protective effect against one ormore prion diseases. For example, prime editing may be used to install aG127S, G127V, M129V, D167G, D167N, N171S, E219K, or P238S protectivemutation in PRNP (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). Instill other embodiments, the protective mutation may be any alternateamino acid installed at G127, G127, M129, D167, D167, N171, E219, orP238 in PRNP (relative to PRNP of NP_000302.1) (SEQ ID NO: 291).

In particular embodiments, prime editing may be used to install a G127Vprotective mutation in PRNP, as illustrated in FIG. 27 and discussed inExample 5.

In another embodiment, prime editing may be used to install an E219Kprotective mutation in PRNP.

The PRNP protein and the protective mutation site are conserved inmammals, so in addition to treating human disease it could also be usedto generate cows and sheep that are immune to prion disease, or evenhelp cure wild populations of animals that are suffering from priondisease. Prime editing has already been used to achieve ˜25%installation of a naturally occurring protective allele in human cells,and previous mouse experiments indicate that this level of installationis sufficient to cause immunity from most prion diseases. This method isthe first and potentially only current way to install this allele withsuch high efficiency in most cell types. Another possible strategy fortreatment is to use prime editing to reduce or eliminate the expressionof PRNP by installing an early stop codon in the gene.

Using the principles described herein for PEgRNA design, appropriatePEgRNAs may be designed for installing desired protective mutations, orfor removing prion disease-associated mutations from PRNP. For example,the below list of PEgRNAs can be used to install the G127V protectiveallele and the E219K protective allele in human PRNP, as well as theG127V protective allele in PRNP of various animals.

[9] Pharmaceutical Compositions

Other aspects of the present disclosure relate to pharmaceuticalcompositions comprising any of the various components of the primeediting system described herein (e.g., including, but not limited to,the napDNAbps, reverse transcriptases, fusion proteins (e.g., comprisingnapDNAbps and reverse transcriptases), extended guide RNAs, andcomplexes comprising fusion proteins and extended guide RNAs, as well asaccessory elements, such as second strand nicking components and 5′endogenous DNA flap removal endonucleases for helping to drive the primeediting process towards the edited product formation).

The term “pharmaceutical composition”, as used herein, refers to acomposition formulated for pharmaceutical use. In some embodiments, thepharmaceutical composition further comprises a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical compositioncomprises additional agents (e.g. for specific delivery, increasinghalf-life, or other therapeutic compounds).

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thecompound from one site (e.g., the delivery site) of the body, to anothersite (e.g., organ, tissue or portion of the body). A pharmaceuticallyacceptable carrier is “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to the tissueof the subject (e.g., physiologically compatible, sterile, physiologicpH, etc.). Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

In some embodiments, the pharmaceutical composition is formulated fordelivery to a subject, e.g., for gene editing. Suitable routes ofadministrating the pharmaceutical composition described herein include,without limitation: topical, subcutaneous, transdermal, intradermal,intralesional, intraarticular, intraperitoneal, intravesical,transmucosal, gingival, intradental, intracochlear, transtympanic,intraorgan, epidural, intrathecal, intramuscular, intravenous,intravascular, intraosseous, periocular, intratumoral, intracerebral,and intracerebroventricular administration.

In some embodiments, the pharmaceutical composition described herein isadministered locally to a diseased site (e.g., tumor site). In someembodiments, the pharmaceutical composition described herein isadministered to a subject by injection, by means of a catheter, by meansof a suppository, or by means of an implant, the implant being of aporous, non-porous, or gelatinous material, including a membrane, suchas a sialastic membrane, or a fiber.

In other embodiments, the pharmaceutical composition described herein isdelivered in a controlled release system. In one embodiment, a pump maybe used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989,CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In anotherembodiment, polymeric materials can be used. (See, e.g., MedicalApplications of Controlled Release (Langer and Wise eds., CRC Press,Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance (Smolen and Ball eds., Wiley, New York, 1984);Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. Seealso Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Othercontrolled release systems are discussed, for example, in Langer, supra.

In some embodiments, the pharmaceutical composition is formulated inaccordance with routine procedures as a composition adapted forintravenous or subcutaneous administration to a subject, e.g., a human.In some embodiments, pharmaceutical composition for administration byinjection are solutions in sterile isotonic aqueous buffer. Wherenecessary, the pharmaceutical can also include a solubilizing agent anda local anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent. Wherethe pharmaceutical is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the pharmaceutical composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

A pharmaceutical composition for systemic administration may be aliquid, e.g., sterile saline, lactated Ringer's or Hank's solution. Inaddition, the pharmaceutical composition can be in solid forms andre-dissolved or suspended immediately prior to use. Lyophilized formsare also contemplated.

The pharmaceutical composition can be contained within a lipid particleor vesicle, such as a liposome or microcrystal, which is also suitablefor parenteral administration. The particles can be of any suitablestructure, such as unilamellar or plurilamellar, so long as compositionsare contained therein. Compounds can be entrapped in “stabilizedplasmid-lipid particles” (SPLP) containing the fusogenic lipiddioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) ofcationic lipid, and stabilized by a polyethyleneglycol (PEG) coating(Zhang Y. P. et al., Gene Ther. 1999, 6:1438-47). Positively chargedlipids such asN-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or“DOTAP,” are particularly preferred for such particles and vesicles. Thepreparation of such lipid particles is well known. See, e.g., U.S. Pat.Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and4,921,757; each of which is incorporated herein by reference.

The pharmaceutical composition described herein may be administered orpackaged as a unit dose, for example. The term “unit dose” when used inreference to a pharmaceutical composition of the present disclosurerefers to physically discrete units suitable as unitary dosage for thesubject, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required diluent; i.e., carrier, or vehicle.

Further, the pharmaceutical composition can be provided as apharmaceutical kit comprising (a) a container containing a compound ofthe invention in lyophilized form and (b) a second container containinga pharmaceutically acceptable diluent (e.g., sterile water) forinjection. The pharmaceutically acceptable diluent can be used forreconstitution or dilution of the lyophilized compound of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

In another aspect, an article of manufacture containing materials usefulfor the treatment of the diseases described above is included. In someembodiments, the article of manufacture comprises a container and alabel. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. In some embodiments, the containerholds a composition that is effective for treating a disease describedherein and may have a sterile access port. For example, the containermay be an intravenous solution bag or a vial having a stopperpierce-able by a hypodermic injection needle. The active agent in thecomposition is a compound of the invention. In some embodiments, thelabel on or associated with the container indicates that the compositionis used for treating the disease of choice. The article of manufacturemay further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

Kits, Cells, Vectors, and Delivery Kits

The compositions of the present disclosure may be assembled into kits.In some embodiments, the kit comprises nucleic acid vectors for theexpression of the prime editors described herein. In other embodiments,the kit further comprises appropriate guide nucleotide sequences (e.g.,PEgRNAs and second-site gRNAs) or nucleic acid vectors for theexpression of such guide nucleotide sequences, to target the Cas9protein or prime editor to the desired target sequence.

The kit described herein may include one or more containers housingcomponents for performing the methods described herein and optionallyinstructions for use. Any of the kit described herein may furthercomprise components needed for performing the assay methods. Eachcomponent of the kits, where applicable, may be provided in liquid form(e.g., in solution) or in solid form, (e.g., a dry powder). In certaincases, some of the components may be reconstitutable or otherwiseprocessible (e.g., to an active form), for example, by the addition of asuitable solvent or other species (for example, water), which may or maynot be provided with the kit.

In some embodiments, the kits may optionally include instructions and/orpromotion for use of the components provided. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the disclosure. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals or biological products, which can alsoreflect approval by the agency of manufacture, use or sale for animaladministration. As used herein, “promoted” includes all methods of doingbusiness including methods of education, hospital and other clinicalinstruction, scientific inquiry, drug discovery or development, academicresearch, pharmaceutical industry activity including pharmaceuticalsales, and any advertising or other promotional activity includingwritten, oral and electronic communication of any form, associated withthe disclosure. Additionally, the kits may include other componentsdepending on the specific application, as described herein.

The kits may contain any one or more of the components described hereinin one or more containers. The components may be prepared sterilely,packaged in a syringe and shipped refrigerated. Alternatively it may behoused in a vial or other container for storage. A second container mayhave other components prepared sterilely. Alternatively the kits mayinclude the active agents premixed and shipped in a vial, tube, or othercontainer.

The kits may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kits maybe sterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kits may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration,etc. Some aspects of this disclosure provide kits comprising a nucleicacid construct comprising a nucleotide sequence encoding the variouscomponents of the prime editing system described herein (e.g.,including, but not limited to, the napDNAbps, reverse transcriptases,polymerases, fusion proteins (e.g., comprising napDNAbps and reversetranscriptases (or more broadly, polymerases), extended guide RNAs, andcomplexes comprising fusion proteins and extended guide RNAs, as well asaccessory elements, such as second strand nicking components (e.g.,second strand nicking gRNA) and 5′ endogenous DNA flap removalendonucleases for helping to drive the prime editing process towards theedited product formation). In some embodiments, the nucleotidesequence(s) comprises a heterologous promoter (or more than a singlepromoter) that drives expression of the prime editing system components.

Other aspects of this disclosure provide kits comprising one or morenucleic acid constructs encoding the various components of the primeediting system described herein, e.g., the comprising a nucleotidesequence encoding the components of the prime editing system capable ofmodifying a target DNA sequence. In some embodiments, the nucleotidesequence comprises a heterologous promoter that drives expression of theprime editing system components.

Some aspects of this disclosure provides kits comprising a nucleic acidconstruct, comprising (a) a nucleotide sequence encoding a napDNAbp(e.g., a Cas9 domain) fused to a reverse transcriptase and (b) aheterologous promoter that drives expression of the sequence of (a).

Cells

Cells that may contain any of the compositions described herein includeprokaryotic cells and eukaryotic cells. The methods described herein areused to deliver a Cas9 protein or a prime editor into a eukaryotic cell(e.g., a mammalian cell, such as a human cell). In some embodiments, thecell is in vitro (e.g., cultured cell. In some embodiments, the cell isin vivo (e.g., in a subject such as a human subject). In someembodiments, the cell is ex vivo (e.g., isolated from a subject and maybe administered back to the same or a different subject).

Mammalian cells of the present disclosure include human cells, primatecells (e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) ormouse cells (e.g., MC3T3 cells). There are a variety of human celllines, including, without limitation, human embryonic kidney (HEK)cells, HeLa cells, cancer cells from the National Cancer Institute's 60cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap(prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breastcancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells,THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Yhuman neuroblastoma cells (cloned from a myeloma) and Saos-2 (bonecancer) cells. In some embodiments, rAAV vectors are delivered intohuman embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells). Insome embodiments, rAAV vectors are delivered into stem cells (e.g.,human stem cells) such as, for example, pluripotent stem cells (e.g.,human pluripotent stem cells including human induced pluripotent stemcells (hiPSCs)). A stem cell refers to a cell with the ability to dividefor indefinite periods in culture and to give rise to specialized cells.A pluripotent stem cell refers to a type of stem cell that is capable ofdifferentiating into all tissues of an organism, but not alone capableof sustaining full organismal development. A human induced pluripotentstem cell refers to a somatic (e.g., mature or adult) cell that has beenreprogrammed to an embryonic stem cell-like state by being forced toexpress genes and factors important for maintaining the definingproperties of embryonic stem cells (see, e.g., Takahashi and Yamanaka,Cell 126 (4): 663-76, 2006, incorporated by reference herein). Humaninduced pluripotent stem cell cells express stem cell markers and arecapable of generating cells characteristic of all three germ layers(ectoderm, endoderm, mesoderm).

Additional non-limiting examples of cell lines that may be used inaccordance with the present disclosure include 293-T, 293-T, 3T3, 4T1,721, 9L, A-549, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, ALC,B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C2C12,C3H-10T1/2, C6, C6/36, Cal-27, CGR8, CHO, CML T1, CMT, COR-L23,COR-L23/5010, COR-L23/CPR, COR-L23/R23, COS-7, COV-434, CT26, D17, DH82,DU145, DuCaP, E14Tg2a, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299,H69, HB54, HB55, HCA2, Hepa1c1c7, High Five cells, HL-60, HMEC, HT-29,HUVEC, J558L cells, Jurkat, JY cells, K562 cells, KCL22, KG1, Ku812,KYO1, LNCap, Ma-Mel 1, 2, 3 . . . 48, MC-38, MCF-10A, MCF-7, MDA-MB-231,MDA-MB-435, MDA-MB-468, MDCK II, MG63, MONO-MAC 6, MOR/0.2R, MRC5,MTD-1A, MyEnd, NALM-1, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20,NCI-H69/LX4, NIH-3T3, NW-145, OPCN/OPCT Peer, PNT-1A/PNT 2, PTK2, Raji,RBL cells, RenCa, RIN-5F, RMA/RMAS, S2, Saos-2 cells, Sf21, Sf9, SiHa,SKBR3, SKOV-3, T-47D, T2, T84, THP1, U373, U87, U937, VCaP, WM39, WT-49,X63, YAC-1 and YAR cells.

Some aspects of this disclosure provide cells comprising any of theconstructs disclosed herein. In some embodiments, a host cell istransiently or non-transiently transfected with one or more vectorsdescribed herein. In some embodiments, a cell is transfected as itnaturally occurs in a subject. In some embodiments, a cell that istransfected is taken from a subject. In some embodiments, the cell isderived from cells taken from a subject, such as a cell line. A widevariety of cell lines for tissue culture are known in the art. Examplesof cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT,mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa,MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24,J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1,SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21,DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS,COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouseembryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts;10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis,A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B,bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7,CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr −/−, COR-L23, COR-L23/CPR,COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82,DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69,HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat,JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48,MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10,NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT celllines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9,SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Verocells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.

Cell lines are available from a variety of sources known to those withskill in the art (see, e.g., the American Type Culture Collection (ATCC)(Manassas, Va.)). In some embodiments, a cell transfected with one ormore vectors described herein is used to establish a new cell linecomprising one or more vector-derived sequences. In some embodiments, acell transiently transfected with the components of a CRISPR system asdescribed herein (such as by transient transfection of one or morevectors, or transfection with RNA), and modified through the activity ofa CRISPR complex, is used to establish a new cell line comprising cellscontaining the modification but lacking any other exogenous sequence. Insome embodiments, cells transiently or non-transiently transfected withone or more vectors described herein, or cell lines derived from suchcells are used in assessing one or more test compounds.

Vectors

Some aspects of the present disclosure relate to using recombinant virusvectors (e.g., adeno-associated virus vectors, adenovirus vectors, orherpes simplex virus vectors) for the delivery of the prime editors orcomponents thereof described herein, e.g., the split Cas9 protein or asplit nucleobase prime editors, into a cell. In the case of a split-PEapproach, the N-terminal portion of a PE fusion protein and theC-terminal portion of a PE fusion are delivered by separate recombinantvirus vectors (e.g., adeno-associated virus vectors, adenovirus vectors,or herpes simplex virus vectors) into the same cell, since thefull-length Cas9 protein or prime editors exceeds the packaging limit ofvarious virus vectors, e.g., rAAV (˜4.9 kb).

Thus, in one embodiment, the disclosure contemplates vectors capable ofdelivering split prime editor fusion proteins, or split componentsthereof. In some embodiments, a composition for delivering the splitCas9 protein or split prime editor into a cell (e.g., a mammalian cell,a human cell) is provided. In some embodiments, the composition of thepresent disclosure comprises: (i) a first recombinant adeno-associatedvirus (rAAV) particle comprising a first nucleotide sequence encoding aN-terminal portion of a Cas9 protein or prime editor fused at itsC-terminus to an intein-N; and (ii) a second recombinantadeno-associated virus (rAAV) particle comprising a second nucleotidesequence encoding an intein-C fused to the N-terminus of a C-terminalportion of the Cas9 protein or prime editor. The rAAV particles of thepresent disclosure comprise a rAAV vector (i.e., a recombinant genome ofthe rAAV) encapsidated in the viral capsid proteins.

In some embodiments, the rAAV vector comprises: (1) a heterologousnucleic acid region comprising the first or second nucleotide sequenceencoding the N-terminal portion or C-terminal portion of a split Cas9protein or a split prime editor in any form as described herein, (2) oneor more nucleotide sequences comprising a sequence that facilitatesexpression of the heterologous nucleic acid region (e.g., a promoter),and (3) one or more nucleic acid regions comprising a sequence thatfacilitate integration of the heterologous nucleic acid region(optionally with the one or more nucleic acid regions comprising asequence that facilitates expression) into the genome of a cell. In someembodiments, viral sequences that facilitate integration compriseInverted Terminal Repeat (ITR) sequences. In some embodiments, the firstor second nucleotide sequence encoding the N-terminal portion orC-terminal portion of a split Cas9 protein or a split prime editor isflanked on each side by an ITR sequence. In some embodiments, thenucleic acid vector further comprises a region encoding an AAV Repprotein as described herein, either contained within the region flankedby ITRs or outside the region. The ITR sequences can be derived from anyAAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or can be derivedfrom more than one serotype. In some embodiments, the ITR sequences arederived from AAV2 or AAV6.

Thus, in some embodiments, the rAAV particles disclosed herein compriseat least one rAAV2 particle, rAAV6 particle, rAAV8 particle, rPHP.Bparticle, rPHP.eB particle, or rAAV9 particle, or a variant thereof. Inparticular embodiments, the disclosed rAAV particles are rPHP.Bparticles, rPHP.eB particles, rAAV9 particles.

ITR sequences and plasmids containing ITR sequences are known in the artand commercially available (see, e.g., products and services availablefrom Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA;Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; andGene delivery to skeletal muscle results in sustained expression andsystemic delivery of a therapeutic protein. Kessler P D, Podsakoff G M,Chen X, McQuiston S A, Colosi P C, Matelis L A, Kurtzman G J, Byrne B J.Proc Natl Acad Sci USA. 1996 Nov. 26; 93(24):14082-7; and Curtis A.Machida. Methods in Molecular Medicine™. Viral Vectors for Gene TherapyMethods and Protocols. 10.1385/1-59259-304-6:201 © Humana Press Inc.2003. Chapter 10. Targeted Integration by Adeno-Associated Virus.Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard JudeSamulski; U.S. Pat. Nos. 5,139,941 and 5,962,313, all of which areincorporated herein by reference).

In some embodiments, the rAAV vector of the present disclosure comprisesone or more regulatory elements to control the expression of theheterologous nucleic acid region (e.g., promoters, transcriptionalterminators, and/or other regulatory elements). In some embodiments, thefirst and/or second nucleotide sequence is operably linked to one ormore (e.g., 1, 2, 3, 4, 5, or more) transcriptional terminators.Non-limiting examples of transcriptional terminators that may be used inaccordance with the present disclosure include transcription terminatorsof the bovine growth hormone gene (bGH), human growth hormone gene(hGH), SV40, CW3, ϕ, or combinations thereof. The efficiencies ofseveral transcriptional terminators have been tested to determine theirrespective effects in the expression level of the split Cas9 protein orthe split prime editor. In some embodiments, the transcriptionalterminator used in the present disclosure is a bGH transcriptionalterminator. In some embodiments, the rAAV vector further comprises aWoodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).In certain embodiments, the WPRE is a truncated WPRE sequence, such as“W3.” In some embodiments, the WPRE is inserted 5′ of thetranscriptional terminator. Such sequences, when transcribed, create atertiary structure which enhances expression, in particular, from viralvectors.

In some embodiments, the vectors used herein may encode the PE fusionproteins, or any of the components thereof (e.g., napDNAbp, linkers, orpolymerases). In addition, the vectors used herein may encode thePEgRNAs, and/or the accessory gRNA for second strand nicking. Thevectors may be capable of driving expression of one or more codingsequences in a cell. In some embodiments, the cell may be a prokaryoticcell, such as, e.g., a bacterial cell. In some embodiments, the cell maybe a eukaryotic cell, such as, e.g., a yeast, plant, insect, ormammalian cell. In some embodiments, the eukaryotic cell may be amammalian cell. In some embodiments, the eukaryotic cell may be a rodentcell. In some embodiments, the eukaryotic cell may be a human cell.Suitable promoters to drive expression in different types of cells areknown in the art. In some embodiments, the promoter may be wild-type. Inother embodiments, the promoter may be modified for more efficient orefficacious expression. In yet other embodiments, the promoter may betruncated yet retain its function. For example, the promoter may have anormal size or a reduced size that is suitable for proper packaging ofthe vector into a virus.

In some embodiments, the promoters that may be used in the prime editorvectors may be constitutive, inducible, or tissue-specific. In someembodiments, the promoters may be a constitutive promoters. Non-limitingexemplary constitutive promoters include cytomegalovirus immediate earlypromoter (CMV), simian virus (SV40) promoter, adenovirus major late(MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumorvirus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter,elongation factor-alpha (EF1a) promoter, ubiquitin promoters, actinpromoters, tubulin promoters, immunoglobulin promoters, a functionalfragment thereof, or a combination of any of the foregoing. In someembodiments, the promoter may be a CMV promoter. In some embodiments,the promoter may be a truncated CMV promoter. In other embodiments, thepromoter may be an EF1a promoter. In some embodiments, the promoter maybe an inducible promoter. Non-limiting exemplary inducible promotersinclude those inducible by heat shock, light, chemicals, peptides,metals, steroids, antibiotics, or alcohol. In some embodiments, theinducible promoter may be one that has a low basal (non-induced)expression level, such as, e.g., the Tet-On® promoter (Clontech). Insome embodiments, the promoter may be a tissue-specific promoter. Insome embodiments, the tissue-specific promoter is exclusively orpredominantly expressed in liver tissue. Non-limiting exemplarytissue-specific promoters include B29 promoter, CD14 promoter, CD43promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAPpromoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter,Nphs1 promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASPpromoter.

In some embodiments, the prime editor vectors (e.g., including anyvectors encoding the prime editor fusion protein and/or the PEgRNAs,and/or the accessory second strand nicking gRNAs) may comprise induciblepromoters to start expression only after it is delivered to a targetcell. Non-limiting exemplary inducible promoters include those inducibleby heat shock, light, chemicals, peptides, metals, steroids,antibiotics, or alcohol. In some embodiments, the inducible promoter maybe one that has a low basal (non-induced) expression level, such as,e.g., the Tet-On® promoter (Clontech).

In additional embodiments, the prime editor vectors (e.g., including anyvectors encoding the prime editor fusion protein and/or the PEgRNAs,and/or the accessory second strand nicking gRNAs) may comprisetissue-specific promoters to start expression only after it is deliveredinto a specific tissue. Non-limiting exemplary tissue-specific promotersinclude B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68promoter, desmin promoter, elastase-1 promoter, endoglin promoter,fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter,ICAM-2 promoter, INF-β promoter, Mb promoter, Nphs1 promoter, OG-2promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

In some embodiments, the nucleotide sequence encoding the PEgRNA (or anyguide RNAs used in connection with prime editing) may be operably linkedto at least one transcriptional or translational control sequence. Insome embodiments, the nucleotide sequence encoding the guide RNA may beoperably linked to at least one promoter. In some embodiments, thepromoter may be recognized by RNA polymerase III (Pol III). Non-limitingexamples of Pol III promoters include U6, HI and tRNA promoters. In someembodiments, the nucleotide sequence encoding the guide RNA may beoperably linked to a mouse or human U6 promoter. In other embodiments,the nucleotide sequence encoding the guide RNA may be operably linked toa mouse or human HI promoter. In some embodiments, the nucleotidesequence encoding the guide RNA may be operably linked to a mouse orhuman tRNA promoter. In embodiments with more than one guide RNA, thepromoters used to drive expression may be the same or different. In someembodiments, the nucleotide encoding the crRNA of the guide RNA and thenucleotide encoding the tracr RNA of the guide RNA may be provided onthe same vector. In some embodiments, the nucleotide encoding the crRNAand the nucleotide encoding the tracr RNA may be driven by the samepromoter. In some embodiments, the crRNA and tracr RNA may betranscribed into a single transcript. For example, the crRNA and tracrRNA may be processed from the single transcript to form adouble-molecule guide RNA. Alternatively, the crRNA and tracr RNA may betranscribed into a single-molecule guide RNA.

In some embodiments, the nucleotide sequence encoding the guide RNA maybe located on the same vector comprising the nucleotide sequenceencoding the PE fusion protein. In some embodiments, expression of theguide RNA and of the PE fusion protein may be driven by theircorresponding promoters. In some embodiments, expression of the guideRNA may be driven by the same promoter that drives expression of the PEfusion protein. In some embodiments, the guide RNA and the PE fusionprotein transcript may be contained within a single transcript. Forexample, the guide RNA may be within an untranslated region (UTR) of theCas9 protein transcript. In some embodiments, the guide RNA may bewithin the 5′ UTR of the PE fusion protein transcript. In otherembodiments, the guide RNA may be within the 3′ UTR of the PE fusionprotein transcript. In some embodiments, the intracellular half-life ofthe PE fusion protein transcript may be reduced by containing the guideRNA within its 3′ UTR and thereby shortening the length of its 3′ UTR.In additional embodiments, the guide RNA may be within an intron of thePE fusion protein transcript. In some embodiments, suitable splice sitesmay be added at the intron within which the guide RNA is located suchthat the guide RNA is properly spliced out of the transcript. In someembodiments, expression of the Cas9 protein and the guide RNA in closeproximity on the same vector may facilitate more efficient formation ofthe CRISPR complex.

The prime editor vector system may comprise one vector, or two vectors,or three vectors, or four vectors, or five vector, or more. In someembodiments, the vector system may comprise one single vector, whichencodes both the PE fusion protein and PEgRNA. In other embodiments, thevector system may comprise two vectors, wherein one vector encodes thePE fusion protein and the other encodes the PEgRNA. In additionalembodiments, the vector system may comprise three vectors, wherein thethird vector encodes the second strand nicking gRNA used in the hereinmethods.

In some embodiments, the composition comprising the rAAV particle (inany form contemplated herein) further comprises a pharmaceuticallyacceptable carrier. In some embodiments, the composition is formulatedin appropriate pharmaceutical vehicles for administration to human oranimal subjects.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

Delivery Methods

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a base editor as described herein in combination with(and optionally complexed with) a guide sequence is delivered to a cell.

Exemplary delivery strategies are described herein elsewhere, whichinclude vector-based strategies, PE ribonucleoprotein complex delivery,and delivery of PE by mRNA methods.

In some embodiments, the method of delivery provided comprisesnucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA.

Exemplary methods of delivery of nucleic acids include lipofection,nucleofection, electoporation, stable genome integration (e.g.,piggybac), microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™,Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationicand neutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Feigner, WO 91/17424; WO91/16024. Delivery may be to cells (e.g. in vitro or ex vivoadministration) or target tissues (e.g. in vivo administration).Delivery may be achieved through the use of RNP complexes.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

In other embodiments, the method of delivery and vector provided hereinis an RNP complex. RNP delivery of fusion proteins markedly increasesthe DNA specificity of base editing. RNP delivery of fusion proteinsleads to decoupling of on- and off-target DNA editing. RNP deliveryablates off-target editing at non-repetitive sites while maintainingon-target editing comparable to plasmid delivery, and greatly reducesoff-target DNA editing even at the highly repetitive VEGFA site 2. SeeRees, H. A. et al., Improving the DNA specificity and applicability ofbase editing through protein engineering and protein delivery, Nat.Commun. 8, 15790 (2017), U.S. Pat. No. 9,526,784, issued Dec. 27, 2016,and U.S. Pat. No. 9,737,604, issued Aug. 22, 2017, each of which isincorporated by reference herein.

Additional methods for the delivery of nucleic acids to cells are knownto those skilled in the art. See, for example, US 2003/0087817,incorporated herein by reference.

Other aspects of the present disclosure provide methods of deliveringthe prime editor constructs into a cell to form a complete andfunctional prime editor within a cell. For example, in some embodiments,a cell is contacted with a composition described herein (e.g.,compositions comprising nucleotide sequences encoding the split Cas9 orthe split prime editor or AAV particles containing nucleic acid vectorscomprising such nucleotide sequences). In some embodiments, thecontacting results in the delivery of such nucleotide sequences into acell, wherein the N-terminal portion of the Cas9 protein or the primeeditor and the C-terminal portion of the Cas9 protein or the primeeditor are expressed in the cell and are joined to form a complete Cas9protein or a complete prime editor.

It should be appreciated that any rAAV particle, nucleic acid moleculeor composition provided herein may be introduced into the cell in anysuitable way, either stably or transiently. In some embodiments, thedisclosed proteins may be transfected into the cell. In someembodiments, the cell may be transduced or transfected with a nucleicacid molecule. For example, a cell may be transduced (e.g., with a virusencoding a split protein), or transfected (e.g., with a plasmid encodinga split protein) with a nucleic acid molecule that encodes a splitprotein, or an rAAV particle containing a viral genome encoding one ormore nucleic acid molecules. Such transduction may be a stable ortransient transduction. In some embodiments, cells expressing a splitprotein or containing a split protein may be transduced or transfectedwith one or more guide RNA sequences, for example in delivery of a splitCas9 (e.g., nCas9) protein. In some embodiments, a plasmid expressing asplit protein may be introduced into cells through electroporation,transient (e.g., lipofection) and stable genome integration (e.g.,piggybac) and viral transduction or other methods known to those ofskill in the art.

In certain embodiments, the compositions provided herein comprise alipid and/or polymer. In certain embodiments, the lipid and/or polymeris cationic. The preparation of such lipid particles is well known. See,e.g. U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951;4,920,016; 4,921,757; and 9,737,604, each of which is incorporatedherein by reference.

The guide RNA sequence may be 15-100 nucleotides in length and comprisea sequence of at least 10, at least 15, or at least 20 contiguousnucleotides that is complementary to a target nucleotide sequence. Theguide RNA may comprise a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40contiguous nucleotides that is complementary to a target nucleotidesequence. The guide RNA may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

In some embodiments, the target nucleotide sequence is a DNA sequence ina genome, e.g. a eukaryotic genome. In certain embodiments, the targetnucleotide sequence is in a mammalian (e.g. a human) genome.

The compositions of this disclosure may be administered or packaged as aunit dose, for example. The term “unit dose” when used in reference to apharmaceutical composition of the present disclosure refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent, i.e., a carrier or vehicle.

Treatment of a disease or disorder includes delaying the development orprogression of the disease, or reducing disease severity. Treating thedisease does not necessarily require curative results.

As used therein, “delaying” the development of a disease means to defer,hinder, slow, retard, stabilize, and/or postpone progression of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individuals being treated. A method that“delays” or alleviates the development of a disease, or delays the onsetof the disease, is a method that reduces probability of developing oneor more symptoms of the disease in a given time frame and/or reducesextent of the symptoms in a given time frame, when compared to not usingthe method. Such comparisons are typically based on clinical studies,using a number of subjects sufficient to give a statisticallysignificant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset.

As used herein “onset” or “occurrence” of a disease includes initialonset and/or recurrence. Conventional methods, known to those ofordinary skill in the art of medicine, can be used to administer theisolated polypeptide or pharmaceutical composition to the subject,depending upon the type of disease to be treated or the site of thedisease.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES Example 1. Prime Editing (PE) for Installing Precise NucleotideChanges in the Genome

The objective is to develop a transformative genome editing technologyfor precise and general installation of single nucleotide changes inmammalian genomes. This technology would allow investigators to studythe effects of single nucleotide variations in virtually any mammaliangene, and potentially enable therapeutic interventions for correctingpathogenic point mutations in human patients.

Adoption of the clustered regularly interspaced short palindromic repeat(CRISPR) system for genome editing has revolutionized the lifesciences¹⁻³. Although gene disruption using CRISPR is now routine, theprecise installation of single nucleotide edits remains a majorchallenge, despite being necessary for studying or correcting a largenumber of disease-causative mutations. Homology directed repair (HDR) iscapable of achieving such edits, but suffers from low efficiency (often<5%), a requirement for donor DNA repair templates, and deleteriouseffects of double-stranded DNA break (DSB) formation. Recently, the Liulaboratory developed base editing, which achieves efficient singlenucleotide editing without DSBs. Base editors (BEs) combine the CRISPRsystem with base-modifying deaminase enzymes to convert target C·G orA·T base pairs to A·T or G·C, respectively⁴⁻⁶. Although already widelyused by researchers worldwide (>5,000 Liu lab BE constructs distributedby Addgene), current BEs enable only four of the twelve possible basepair conversions and are unable to correct small insertions ordeletions. Moreover, the targeting scope of base editing is limited bythe editing of non-target C or A bases adjacent to the target base(“bystander editing”) and by the requirement that a PAM sequence exist15±2 bp from the target base. Overcoming these limitations wouldtherefore greatly broaden the basic research and therapeuticapplications of genome editing.

Here, it is proposed to develop a new precision editing approach thatoffers many of the benefits of base editing—namely, avoidance of doublestrand breaks and donor DNA repair templates—while overcoming its majorlimitations. To achieve this ambitious goal, it is aimed to directlyinstall edited DNA strands at target genomic sites using target-primedreverse transcription (TPRT). In the design discussed herein, CRISPRguide RNA (gRNA) will be engineered to carry a template encodingmutagenic DNA strand synthesis, to be executed by an associated reversetranscriptase (RT) enzyme. The CRISPR nuclease (Cas9)-nicked target siteDNA will serve as the primer for reverse transcription, allowing fordirect incorporation of any desired nucleotide edit.

Section 1

Establish guide RNA-templated reverse transcription of mutagenic DNAstrands. Prior studies have shown that, following DNA cleavage but priorto complex dissociation, Cas9 releases the non-target DNA strand toexpose a free 3′ terminus. It is hypothesized that this DNA strand isaccessible to extension by polymerase enzymes, and that gRNAs can beengineered through extension of their 5′ or 3′ terminus to serve astemplates for DNA synthesis. In preliminary in vitro studies, it wasestablished that nicked DNA strands within Cas9:gRNA-bound complexes canindeed prime reverse transcription using the bound gRNA as a template(RT enzyme in trans). Next, different gRNA linkers, primer bindingsites, and synthesis templates will be explored to determine optimaldesign rules in vitro. Then, different RT enzymes, acting in trans or asfusions to Cas9, will be evaluated in vitro. Finally, engineered gRNAdesigns will be identified that retain efficient binding and cuttingactivity in cells. Successful demonstration of this aim will provide afoundation for carrying out mutagenic strand synthesis in cells.

Section 2

Establish prime editing in human cells. Based on DNA processing andrepair mechanisms, it is hypothesized that mutagenic DNA strands (singlestranded flaps) can be used to direct specific and efficient editing oftarget nucleotides. In encouraging preliminary studies, feasibility forthis strategy was established by demonstrating editing with modelplasmid substrates containing mutagenic flaps. Concurrent with Aim 1,repair outcomes will be further evaluated by systematically varying themutagenic flap's length, sequence composition, target nucleotideidentity, and 3′ terminus. Small 1 to 3 nucleotide insertions anddeletions will also be tested. In parallel, and building from Aim 1,Cas9-RT architectures will be evaluated, including fusion proteins andnon-covalent recruitment strategies. Cas9-RT architectures and extendedgRNAs will be assayed for cellular editing at multiple target sites inthe human genome, and will then be optimized for high efficiency. Ifsuccessful, this aim would immediately establish TPRT genome editing(i.e., prime editing) for basic science applications.

Section 3

Achieve site-specific editing of pathogenic mutations in cultured humancells. The potential generality of this technology could enable editingof transversion mutations and indels that are not currently correctableby BEs. Guided by the results of Aim 1 and Aim 2, pathogenictransversion mutations will be targeted in cultured human cells,including the sickle cell disease founder mutation in beta globin(requires an A·T to T·A transversion to correct) and the most prevalentWilson's disease mutation in ATP7B (requires a G·C to T·A transversionto correct). The correction of small insertion and deletion mutationswill also be examined, including the 3-nucleotide ΔF508 deletion in CFTRthat causes cystic fibrosis. If successful, this would lay thefoundation for developing powerful therapeutic approaches that addressthese important human diseases.

Approach

The objective is to develop a genome editing strategy that directlyinstalls point mutations at targeted genomic sites. In the technologydevelopment phase, efforts will focus on protein and RNA engineering toincorporate TPRT functionality into the CRISPR/Cas system. In vitroassays will be used to carefully probe the function of each step ofTPRT, building from the ground up (Aim 1). The second focus area willevaluate editing outcomes in mammalian cells using a combination ofmodel substrates and engineered CRISPR/Cas systems (Aim 2). Finally, theapplication phase will use the technology to correct mutations that havebeen intractable to genome editing by other methods (Aim 3).

The general editing design is shown in FIGS. 1A-1B. Cas9 nickasescontain inactivating mutations to the HNH nuclease domain (Spy Cas9H840A or N863A), restricting DNA cleavage to the PAM containing strand(non-target strand). Guide RNAs (gRNAs) are engineered to contain atemplate for reverse transcription (designs detailed on slide 5). Shownis a 5′ extension of the gRNA, but 3′ extensions can also beimplemented. The Cas9 nickase is fused to a reverse transcriptase (RT)enzyme, either through the C-terminus or N-terminus. The gRNA:Cas9-RTcomplex targets the DNA region of interest and forms an R loop afterdisplacing the non-target strand. Cas9 nicks the non-target DNA strand.Release of the nicked strand exposes a free 3′-OH terminus that iscompetent to prime reverse transcription using the extended gRNA as atemplate. This DNA synthesis reaction is carried out by the fused RTenzyme. The gRNA template encodes a DNA sequence that is homologous tothe original DNA duplex, with the exception of the nucleotide that istargeted for editing. The product of reverse transcription is a singlestranded DNA flap that encodes the desired edit. This flap, whichcontains a free 3′ terminus, can equilibrate with the adjacent DNAstrand, resulting in a 5′ flap species. The latter species ishypothesized to serve as an efficient substrate for FEN1 (flapendonuclease 1), an enzyme that naturally excises 5′ flaps from Okazakifragments during lagging strand DNA synthesis, and removes 5′ flapsfollowing strand displacement synthesis that occurs during long-patchbase excision repair. Ligation of the nicked DNA produces a mismatchedbase pair. This intermediate could either undergo reversion to theoriginal base pair or conversion to the desired edited base pair viamismatch repair (MMR) processes. Alternatively, semiconservative DNAreplication could give rise to one copy each of the reversion and edit.

1. Establish Guide RNA-Templated Reverse Transcription of Mutagenic DNAStrands.

Background and Rationale

In the proposed genome editing strategy, the Cas9-nicked non-target DNAstrand (PAM-containing strand that forms the R-loop) acts as the primerfor DNA synthesis. It is hypothesized that this is possible based onseveral pieces of biochemical and structural data. Nuclease protectionexperiments³², crystallographic studies³³, and base editingwindows^(4,24) have demonstrated a large degree of flexibility anddisorder for the non-target strand nucleotides −20 through −10 withinthe so-called R-loop of the Cas9-bound complex (numbering indicatesdistance 5′ from first PAM nucleotide). Moreover, the PAM-distal portionof the cleaved non-target strand can be displaced from tightly boundternary complexes when complementary ssDNA is added in trans²⁰. Thesestudies support that the non-target strand is highly flexible, isaccessible to enzymes, and that after nicking, the 3′ terminus of thePAM-distal fragment is released prior to Cas9 dissociation. Furthermore,it is hypothesized that gRNAs can be extended to template DNA synthesis.Prior studies have shown that gRNAs for SpCas9, SaCas9, and LbCas12a(formerly Cpf1) tolerate gRNA extensions with RNA aptamers³⁴,ligand-inducible self-cleaving ribozymes³⁵, and long non-coding RNAs³⁶.This literature establishes precedent for two major features that willbe exploited. In assessing this strategy, several CRISPR-Cas systemswill be evaluated in conjunction with 5′ and 3′ extended gRNA designsusing a combination of in vitro and cellular assays (FIGS. 2A-2C).

Designs for engineered gRNAs for TRT editing are shown in FIGS. 3A-3B.DNA synthesis proceeds 5′ to 3′, and thus copies the RNA template in the3′ to 5′ direction. The design for the 5′ extension contains a linkerregion, a primer binding site where the nicked DNA strand anneals, and atemplate for DNA synthesis by reverse transcription. The 3′ extendedgRNA contains a primer binding site and a reverse transcriptiontemplate. In some cases, the 3′ RNA hairpin of the gRNA core is modifiedto match the DNA target sequence, as in vitro experiments showed thatreverse transcription extends ˜3 nucleotides into the gRNA core for the3′ extended gRNA constructs (modification of the hairpin sequenceappears well tolerated so long as compensatory changes are made thatmaintain the hairpin RNA structure). DNA synthesis proceed 5′ to 3′,with nucleotides added to the 3′ OH of the growing DNA strand.

Preliminary Results

Cas9 nicked DNA primes reverse transcription of gRNA templates. Toevaluate the accessibility of the nicked non-target DNA strand, in vitrobiochemical assays were performed using the Cas9 nuclease from S.pyogenes (SpCas9) and Cy5 fluorescently labeled duplex DNA substrates(51 base pairs). First, a series of gRNAs containing 5′ extensions withvarying synthesis template lengths were prepared by in vitrotranscription (overall design shown in FIG. 2B). Electrophoreticmobility shift assays (EMSA) with nuclease dead Cas9 (dCas9) establishedthat 5′ extended gRNAs maintain target binding affinity (data notshown). Next, TPRT activity was tested on pre-nicked Cy5-labeled duplexDNA substrates using dCas9, 5′-extended gRNAs, and Molony-MurineLeukemia Virus (M-MLV) reverse transcriptase (Superscript III). After 1hour of incubation at 37° C., products were evaluated by denaturingpolyacrylamide gel electrophoresis (PAGE) and imaged using Cy5fluorescence (FIG. 4A). Each 5′-extended gRNA variant led to significantproduct formation, with the observed DNA product sizes being consistentwith the length of the extension template (FIG. 4B). Importantly, in theabsence of dCas9, pre-nicked substrates were extended to the full 51-bplength of the DNA substrate, strongly suggesting that the complementaryDNA strand, and not the gRNA, was used as the template for DNA synthesiswhen dCas9 was not present (FIG. 4C). Of note, the system was designedsuch that the newly synthesized DNA strand mirrors the product thatwould be required for target site editing (a homologous strand with asingle nucleotide change). This result establishes that Cas9:gRNAbinding exposes the nicked non-target strand's 3′ end, and that thenon-target strand is accessible to reverse transcription.

Next, non-nicked dsDNA substrates were evaluated using the Cas9(H840A)mutant, which nicks the non-target DNA strand. First, to testCas9(H840A) nickase activity with 5′-extended gRNAs, in vitro cleavageassays were performed as previously described³⁷. Although nicking wasimpaired by comparison to the standard gRNA, appreciable cleavageproducts were formed (FIG. 4D). Importantly, RT products were alsoobserved when TPRT reactions were carried out with 5′-extended gRNAs andCas9(H840A), albeit at lower yields that are likely explained by thedecreased nicking activity (FIG. 4D). This result establishes that5′-extended gRNA:Cas9(H840A) complexes can nick DNA and template reversetranscription.

Finally, 3′ gRNA extensions were evaluated for Cas9(H840A) nicking andTPRT. By comparison to 5′-extended gRNAs, DNA cleavage by 3′-extendedgRNAs was not impaired to any detectable extent compared to the standardgRNA. Significantly, 3′-extended gRNA templates also supported efficientreverse transcription with both pre-nicked and intact duplex DNAsubstrates when M-MLV RT was supplied in trans (FIG. 4E). Surprisingly,only a single product was observed for 3′-extended templates, indicatingthat reverse transcription terminates at a specific location along thegRNA scaffold. Homopolymer tailing of the product with terminaltransferase followed by Klenow extension and Sanger sequencing revealedthat the full gRNA synthesis template was copied in addition to theterminal 3 nucleotides of the gRNA core. In the future, the flapterminus will be reprogrammed by modifying the terminal gRNAsequence^(38,39). This result demonstrates that 3′-extended gRNAs canserve as efficient nuclease targeting guides and can template reversetranscription.

Cas9-TPRT uses nicked DNA and gRNA in cis. Dual color experiments wereused to determine if the RT reaction preferentially occurs with the gRNAin cis (bound in the same complex) (see FIG. 8 ). Two separateexperiments were conducted for 5′-extended and 3′-extended gRNAs. For agiven experiment, ternary complexes of dCas9, gRNA, and DNA substratewere formed in separate tubes. In one tube, the gRNA encodes a long RTproduct and the DNA substrate is labeled with Cy3; in the other, thegRNA encodes a short RT product and the DNA substrate is labeled withCy5. After short incubations, the complexes were mixed and then treatedwith RT enzyme and dNTPs. Products were separated by urea-denaturingPAGE and visualized by fluorescence in the Cy3 and Cy5 channels.Reaction products were found to preferentially form using the gRNAtemplate that was pre-complexed with the DNA substrate, indicating thatthe RT reaction likely can occur in cis. This results supports that asingle Cas9:gRNA complex can target a DNA site and template reversetranscription of a mutagenic DNA strand.

Testing TPRT with Other Cas Systems

Similar experiments to those presented in the previous sections will becarried out using other Cas systems, including Cas9 from S. aureus andCas12a from L. bacterium (see FIGS. 2A-2C). If TPRT can also bedemonstrated for these Cas variants, the potential editing scope andlikelihood of overall success in cells would increase.

Testing TPRT with RT-Cas9fusion Proteins

A series of commercially available or purifiable RT enzymes will firstbe evaluated in trans for TPRT activity. In addition to the alreadytested RT from M-MLV, the RT from Avian Myeloblastosis Virus (AMV), theGeobacillus stearothermophilus Group II Intron (GsI-IIC)^(41,42), andthe Eubacterium rectale group II intron (Eu.re.I2)^(43,44) will beevaluated. Significantly, the latter two RTs perform TPRT in theirnatural biological contexts. Where relevant, RNAse inactivatingmutations and other potentially beneficial RT enzyme modifications willbe tested. Once functional RTs are identified when supplied in trans,each will be evaluated as a fusion protein to Cas9 variants. BothN-terminus and C-terminus fusion orientations will be tested, along withvarious linker lengths and architectures. Kinetic time courseexperiments will be used to determine whether TPRT can occur using theRT enzyme in cis. If an RT-Cas9 fusion architecture can be constructedthat allows for efficient TPRT chemistry, this will greatly increase thelikelihood of functional editing in the context of a cell.

Cas9 Targeting with Engineered gRNAs in Cells

Candidate engineered gRNAs developed in the previous sub-aims will beevaluated in human cell culture experiments (HEK293) to confirm Cas9targeting efficiency. Using established indel formation assays employingwild type SpCas9⁴⁵, engineered gRNAs will be compared side-by-side withstandard gRNAs across 5 or more sites in the human genome. Genomeediting efficiency will be characterized by amplicon sequencing inmultiplex using the Illumina MiSeq platform housed in the laboratory. Itis anticipated that results from this and the preceding sections willgenerate insights that inform subsequent iterations of thedesign-build-test cycle, where gRNAs can be optimized for bothtemplating reverse transcription and efficient Cas9 targeting in cells.

Results of in vitro validations are shown in FIGS. 5-7 . In vitroexperiments demonstrated that the nicked non-target DNA strand isflexible and available for priming DNA synthesis, and that the gRNAextension can serve as a template for reverse transcription (see FIG. 5). This set of experiments used 5′-extended gRNAs (designed as shown inFIGS. 3A-3B) with varying length synthesis templates (listed to theleft). Fluorescently labeled (Cy5) DNA targets were used as substrates,and were pre-nicked in this set of experiments. The Cas9 used in theseexperiments is catalytically dead Cas9 (dCas9), so cannot cut DNA butcan still bind efficiently. Superscript III, a commercial RT derivedfrom the Moloney-Murine Leukemia Virus (M-MLV), was supplied in trans.First, dCas9:gRNA complexes were formed from purified components. Then,the fluorescently labeled DNA substrate was added along with dNTPs andthe RT enzyme. After 1 hour of incubation at 37 C, the reaction productswere analyzed by denaturing urea-polyacrylamide gel electrophoresis(PAGE). The gel image shows extension of the original DNA strand tolengths that are consistent with the length of the reverse transcriptiontemplate. Of note, reactions carried out in the absence of dCas9produced DNA products of length 51 nucleotides, regardless of the gRNAused. This product corresponds to use of the complementary DNA strand asthe template for DNA synthesis and not the RNA (data not shown). Thus,Cas9 binding is required for directing DNA synthesis to the RNAtemplate. This set of in vitro experiments closely parallels those shownin FIG. 5 , except that the DNA substrate is not pre-nicked, and a Cas9nickase (SpyCas9 H840A mutant) is used. As shown in the gel, the nickaseefficiently cleaves the DNA strand when the standard gRNA is used(gRNA_0, lane 3). Multiple cleavage products are observed, consistentwith prior biochemical studies of SpyCas9. The 5′ extension impairsnicking activity (lanes 4-8), but some RT product is still observed.FIG. 7 shows that 3′ extensions support DNA synthesis and do notsignificantly effect Cas9 nickase activity. Pre-nicked substrates (blackarrow) are near-quantitatively converted to RT products when eitherdCas9 or Cas9 nickase is used (lanes 4 and 5). Greater than 50%conversion to the RT product (white arrow) is observed with fullsubstrates (lane 3). To determine the length and sequence of the RTproduct, the product band was excised from the gel, extracted, andsequenced. This revealed that RT extended 3 nucleotides into the gRNAcore's 3′ terminal hairpin. Subsequent experiments (not shown)demonstrated that these three nucleotides could be changed to matchtarget DNA sequences, so long as complementary changes were made thatmaintain the hairpin RNA structure.

Potential Difficulties and Alternatives

(1) RT does not function as a fusion: molecular crowding and/orunfavorable geometries could encumber polymerase extension by Cas9-fusedRT enzymes. First, linker optimization can be tested. Circularlypermutated variants of Cas9, which could re-orient the spatialrelationship between the DNA primer, gRNA, and RT enzyme, will beevaluated. Non-covalent RT recruitment strategies as detailed in Aim 2can be tested. (2) Decreased Cas targeting efficiency by extended gRNAvariants: this is most likely to be an issue for 5′-extended gRNAs.Based on structural data²⁴, Cas9 mutants can be designed and screened toidentify variants with greater tolerance to gRNA extension. In addition,gRNA libraries could be screened in cells for linkers that improvetargeting activity.

Significance

These preliminary results establish that Cas9 nickases and extendedgRNAs can initiate target-primed reverse transcription on bound DNAtargets using a reverse transcriptase supplied in trans. Importantly,Cas9 binding was found to be critically important for product formation.Though perhaps not an absolute requirement for genome editing in cells,further development of the system that incorporates RT enzyme functionin cis would significantly increase the likelihood of success incell-based applications. Achievement of the remaining aspects of thisAim would provide a molecular foundation for carrying out precisiongenome editing in the context of the human genome.

2. Establish Prime Editing in Human Cells.

Background and Rationale

In the proposed strategy, an engineered RT-Cas9:gRNA complex willintroduce mutagenic 3′ DNA flaps at genomic target sites. It ishypothesized that mutagenic 3′ flaps containing a single mismatch willbe incorporated by the DNA repair machinery through energeticallyaccessible equilibration with adjacent 5′ flaps, which would bepreferentially removed (FIGS. 1C-1D). The DNA replication and repairmachineries encounter 5′ ssDNA flaps when processing Okazaki fragments⁴⁶and during long-patch base excision repair (LP-BER)⁴⁷. 5′ flaps are thepreferred substrates for the widely expressed flap endonuclease FEN1,which is recruited to DNA repair sites by the homotrimeric sliding clampcomplex PCNA⁴⁸. PCNA also serves as a scaffold for simultaneousrecruitment of other repair factors including the DNA ligase Lig1⁴⁹.Acting as a ‘toolbelt’, PCNA accelerates serial flap cleavage andligation, which is essential to processing the millions of Okazakifragments generated during every cell division^(50,51). Based onresemblance to these natural DNA intermediates, it is hypothesized thatmutagenic strands would be incorporated through equilibration with 5′flaps, followed by coordinated 5′ flap excision and ligation. Mismatchrepair (MMR) should then occur on either strand with equal probability,leading to editing or reversion (FIGS. 1C-1D). Alternatively, DNAreplication could occur first and lead directly to the incorporation ofthe edit in the newly synthesized daughter strand. While the highestexpected yield from this process is 50%, multiple substrate editingattempts could drive the reaction toward completion due to theirreversibility of editing repair.

Preliminary Result

DNA flaps induce site-specific mutagenesis in plasmid model substratesin yeast and HEK cells. To test the proposed editing strategy, studieswere initiated with model plasmid substrates containing mutagenic 3′flaps that resemble the product of TPRT. A dual fluorescent proteinreporter was created that encodes a stop codon between GFP and mCherry.Mutagenic flaps encode a correction to the stop codon (FIG. 9A),enabling mCherry synthesis. Thus, mutagenesis efficiency can bequantified by GFP:mCherry ratios. Plasmid substrates were prepared invitro and introduced into yeast (S. cerevisiae) or human cells (HEK293).High frequency mutagenesis was observed in both systems (FIG. 9B), andisolated yeast colonies contained either the reverted base, the mutatedbase, or a mixture of both products (FIG. 9C). Detection of the lattersuggests that plasmid replication occurred prior to MMR in these cases,and further suggests that flap excision and ligation precede MMR. Thisresult establishes the feasibility of DNA editing using 3′ mutagenicstrands.

Systematic Studies with Model Flap Substrates

Based on the preliminary results described above, a broader spectrum offlap substrates will be evaluated in HEK cells to infer principles ofefficient editing. 3′ ssDNA flaps will be systematically varied todetermine the influence of mismatch pairings, the location of themutagenic nucleotide along the flap, and the identity of the terminalnucleotide (FIG. 9D). Single nucleotide insertions and deletions willalso be tested. Amplicon sequencing will be used to analyze editingprecision. These results will help inform the design of gRNA reversetranscription templates.

In vitro TPRT on plasmid substrates leads to efficient editing outcomes.TPRT reactions developed in Aim 1 were used to induce mutagenesis withina plasmid substrate. The reaction was carried out on circular DNAplasmid substrates (see FIG. 10 ). This rules out the possibility of DNAstrand dissociation as the mechanism for RT extension in the previous invitro experiments. It also allowed for the testing of DNA repair of flapsubstrates in cells. A dual-fluorescent reporter plasmid was constructedfor yeast (S. cerevisiae) expression. This plasmid encodes GFP (greenfluorescent protein) and mCherry (red fluorescent protein) with anintervening stop codon (TGA). Expression of this construct in yeastproduces only GFP. The plasmid was used as a substrate for in vitro TRT[Cas9(H840A) nickase, engineered gRNA, MLV RT enzyme, dNTPS]. The gRNAextension encodes a mutation to the stop codon. The flap strand is usedfor repair of the stop codon and it is anticipated to produce a plasmidthat expresses both GFP and mCherry as a fusion protein. Yeast dual-FPplasmid transformants are shown in FIG. 10 . Transforming the parentplasmid or an in vitro Cas9(H840A) nicked plasmid results in only greenGFP expressing colonies. TRT reaction with 5′-extended or 3′-extendedgRNAs produces a mix of green and yellow colonies. The latter expressboth GFP and mCherry. More yellow colonies are observed with the3′-extended gRNA. A positive control that contains no stop codon isshown as well.

This result establishes that long double stranded substrates can undergoTPRT, and that TPRT products induce editing in eukaryotic cells.

Another experiment similar to the foregoing prime editing experiment wascarried out, but instead of installing a point mutation in the stopcodon, TRT editing installs a single nucleotide insertion (left) ordeletion (right) that repairs a frameshift mutation and allows forsynthesis of downstream mCherry (see FIG. 11 ). Both experiments used 3′extended gRNAs. Individual colonies from the TRT transformations wereselected and analyzed by Sanger sequencing (see FIG. 12 ). Greencolonies contained plasmids with the original DNA sequence, while yellowcolonies contained the precise mutation designed by the TRT editinggRNA. No other point mutations or indels were observed.

Establish Prime Editing in HEK Cells Using RT-Cas9 Architectures

The optimized constructs from previous aims will be adapted formammalian expression and editing at targeted sites in the human genome.Multiple RT enzymes and fusion architectures will be tested, in additionto adjacent targeting with secondary gRNAs (truncated to preventnicking). Non-covalent RT recruitment will also be evaluated using theSun-Tag system⁵² and MS2 aptamer system⁵³. Indel formation assays willbe used to evaluate targeting efficiency with standard gRNAs and RT-Cas9fusions (as above). Then, for each genomic site, extended gRNAs andRT-Cas9 pairs will be assayed for single nucleotide editing. Editingoutcomes will be evaluated with MiSeq.

Initial experiments in HEK cells were performed using Cas9-RT fusions.Editing by components expressed within cells requires a Cas9(H840A)nickase, a reverse transcriptase (expressed as a fusion or supplied intrans), and an engineered gRNA with a 3′ extension (see FIG. 14 ).Preliminary studies indicated that the length of the primer binding sitewithin the gRNA extension was important for increasing the efficiency ofediting in human cells (see FIG. 15 ).

Optimize Prime Editing Parameters in HEK Cells

After identifying Cas9-RT architectures that can perform prime editingin cells, the components and design will be optimized to achieve highefficiency editing. The location and nucleotide identity of the encodedpoint mutation, and the total length of the newly synthesized DNAstrand, will be varied to evaluate editing scope and potentiallimitations. Short insertion and deletion mutations will also beevaluated. Protein expression constructs will be codon optimized. Ifsuccessful, this would establish efficient prime editing in mammaliancells.

Preliminary Result. Additional gRNAs were designed to bring the RTenzyme to a higher local concentration at the editing locus, in theevent that intramolecular reverse transcription by the fused RT enzymewere not possible. These auxiliary guides are truncated at the 5′ end(14-15 nt spacer), which has previously been shown to prevent Cas9cutting but retain binding (see FIG. 16 ). The HEK3 locus was chosen toexplore this strategy.

Potential Difficulties and Alternatives

1) gRNA degradation in cells: if extended gRNA termini are truncated incells, stabilizing secondary structures could be installed, or syntheticgRNAs with stabilizing modifications could be tested. (2) No observedediting in human cells: additional strategies will be explored,including secondary targeting of RT-Cas9 fusions to adjacent genomicsites⁵⁴. In addition, potential directed evolution strategies in E. colior S. cerevisiae could be explored.

Significance

If prime editing could be established in experimental cell lines, thiswould have an immediate impact for basic biomedical research by enablingthe rapid generation and characterization of a large number of pointmutations in human genes. The generality of the method, and itsorthogonal editing window with respect to base editors, would provide anapproach to installing many currently inaccessible mutations. Moreover,if prime editing could be optimized for high efficiency and productpurity, its potential applicability to correcting disease mutations inother human cell types would be significant.

3. Achieve Site-Specific Editing of Pathogenic Mutations in CulturedHuman Cells.

Background and Rationale.

A large number of pathogenic mutations cannot be corrected by currentbase editors due to PAM restrictions, or a need for transversion orindel mutation correction. With prime editing, all transitions andtransversions are theoretically possible, as may be small insertions anddeletions. Moreover, in relation to the PAM, the prime editing window(anticipated −3 to +4) is distinct from that of base editors (−18 to−12) (FIG. 13 ). Mendelian conditions not currently correctable by baseeditors include: (1) the sickle cell disease Glu6Val founder mutation inhemoglobin beta (requires A·T to T·A transversion); (2) the most commonWilson's disease variant His1069Gln in ATP7B (requires G·C to T·Atransversion); and (3) the ΔPhe508 mutation in CFTR that causes cysticfibrosis (requires 3-nucleotide insertion). Each of these targetscontains an appropriately positioned PAM for SpCas9 targeting and primeediting.

Preliminary Results.

T to A Editing in HEK3 Cells is not Achievable by Current Base Editingbut is Achievable by TRPT Editing (See FIGS. 17A-17C).

FIG. 17A shows a graph displaying the % T to A conversion at the targetnucleotide after transfection of components in human embryonic kidney(HEK) cells. This data presents results using an N-terminal fusion ofwild type MLV reverse transcriptase to Cas9(H840A) nickase (32-aminoacid linker). Editing efficiency was improved dramatically when thelength of the primer binding site is extended from 7 nucleotides to 11or 12 nucleotides. Additionally, the auxiliary guide A, which ispositioned just upstream of the editing locus (see FIG. 16 ),significantly improves editing activity, particularly for shorter lengthprimer binding sites. Editing efficiency was quantified by ampliconsequencing using the Illumina MiSeq platform. FIG. 17B also shows % T toA conversion at the target nucleotide after transfection of componentsin human embryonic kidney (HEK) cells, but this data presents resultsusing a C-terminal fusion of the RT enzyme. Here, the auxiliary guide Adoes not have as much of an effect, and editing efficiency is overallhigher. FIG. 17C shows data presenting results using an N-terminalfusion of wild type MLV reverse transcriptase to Cas9(H840A) nickasesimilar to that used in FIG. 17A; however the linker between the MLV RTand Cas9 is 60 amino acids long instead of 32 amino acids.

T to A Editing at HEK3 Site by TRPT Editing Results Displays HighPurity.

FIG. 18 shows the output of sequencing analysis by high-throughputamplicon sequencing. The output displays the most abundant genotypes ofedited cells. Of note, no major indel products are obtained, and thedesired point mutation (T to A) is cleanly installed without bystanderedits. The first sequence shows the reference genotype. The top twoproducts are the starting genotype containing an endogenous polymorphism(G or A). The bottom two products represent the correctly editedgenotypes.

MLV RT Mutants Improve Editing.

Mutant reverse transcriptases, described in Baranauskas, et al(doi:10.1093/protein/gzs034), were tested as C-terminal fusions to theCas9(H840A) nickase for target nucleotide editing in human embryonickidney (HEK) cells. Cas9-RT editor plasmid was co-transfected with aplasmid encoding a 3′-prime editing guide RNA that templates reversetranscription. Editing efficiency at the target nucleotide (stripedbars) is shown alongside indel rates (white bars) in FIG. 19 . WT refersto the wild type MLV RT enzyme. The mutant enzymes (M1 through M4)contain the mutations listed to the right. Editing rates were quantifiedby high throughput sequencing of genomic DNA amplicons.

Complementary Strand Nicking with a Second gRNA Improves Editing.

This experiment evaluates editing efficiency of the target nucleotidewhen a single strand nick is introduced in the complementary DNA strandin proximity to the target nucleotide, with the hypothesis being thatthis would direct mismatch repair to preferentially remove the originalnucleotide and convert the base pair to the desired edit. TheCas9(H840A)-RT editing construct was co-transfected with two guide RNAencoding plasmids, one of which templates the reverse transcriptionreaction, while the other targets the complementary DNA strand fornicking. Nicking at various distances from the target nucleotide wastested (triangles) (see FIG. 20 ). Editing efficiency at the target basepair (striped bars) is shown alongside the indel formation rate (whitebars). The “none” example does not contain a complementary strandnicking guide RNA. Editing rates were quantified by high throughputsequencing of genomic DNA amplicons.

FIG. 21 shows processed high throughput sequencing data showing thedesired T to A transversion mutation and general absence of other majorgenome editing byproducts.

Scope. The potential scope for the new editing technology is shown inFIG. 13 and is compared to deaminase-mediated base editor technologies.Previously developed base editors target a region ˜15±2 bp upstream ofthe PAM. By converting target C or A nucleotides to T or G,respectively, previously developed base editors enable all transitionmutations (A:T to G:C conversions). However, previously developed baseeditors are unable to install transversion mutations (A to T, A to C, Gto T, G to C, T to A, T to G, C to A, C to G). Moreover, if there aremultiple target nucleotides in the editing window, additional undesirededits can result.

The new prime editing technology could theoretically install anynucleotide and base pair conversion, and potentially small insertion anddeletion edits as well. With respect to the PAM, prime editing windowsstart at the site of DNA nicking (3 bases upstream of the PAM) and endat an as-of-yet undetermined position downstream of the PAM. Of note,this editing window is distinct from that of deaminase base editors.Because the TPRT systems performs editing using DNA polymerase enzymes,it potentially has all of their benefits including generality,precision, and fidelity.

Correct Pathogenic Mutations in Patient-Derived Cell Lines.

Cell lines harboring the relevant mutations (sickle cell disease: CD34+hematopoietic stem cells; Wilson's disease: cultured fibroblasts; cysticfibrosis: cultured bronchial epithelia) will be obtained from ATCC, theCoriell Biobank, or collaborating Harvard/Broad affiliate laboratories.Editing efficiency will be evaluated by high throughput sequencing, andthe efficacy of the corrected genotype will be tested using phenotypicassays (hemoglobin HPLC, ATP7B immunostaining, and CFTR membranepotential assays).

Characterize Off-Target Editing Activity.

Potential off-target editing will be screened with established methodssuch as GUIDE-seq⁵⁵ and CIRCLE-seq⁵⁶ using target gRNAs paired with wildtype Cas9. If potential off-targets are identified, these loci will beprobed in TPRT edited cells to identify true off-target editing events.

Potential Difficulties and Alternatives.

(1) Low editing efficiency: prime editor (PE)s may require optimizationfor each target. In this case, gRNA libraries can be tested to identifythe highest functioning variants for specific applications. RT-Casfusion expression and nuclear localization can be optimized. LiposomalRNP delivery could be used to limit off-target editing.

Upcoming Experiments.

Optimization of gRNA designs can be achieved by further exploration ofthe primer binding site length and extension of synthesis template.Testing scope and generality will include different nucleotideconversions, small insertions and deletions, as well as, differentediting positions with respect to PAM, and multiple sites in the humangenome. Optimization of RT component will include exploring mutations inMLV RT to enhance activity (Rnase H inactivation, increaseprimer-template binding affinity, adjustments to processivity), and newRT enzymes (group II intro RTs, other retroviral RTs).

Significance.

Myriad genetic disorders result from single nucleotide changes inindividual genes. Developing the genome editing technology describedhere, and applying it in disease-relevant cell types, would establish afoundation for translation to the clinic. For some diseases, such asSickle Cell Disease, a single point mutation represents the dominantgenotype throughout the population. However, for many other geneticdisorders, a large heterogeneity of different point mutations within asingle gene is observed throughout the patient population, each of whichgives rise to a similar disease phenotype. Therefore, as a generalgenome editing method that could in theory target a large number of suchmutations, this technology could provide enormous potential benefit tomany of these patients and their families. If proof of principle forthese applications could be established in cells, it would establish thefoundation to studies in animal models of disease.

Advantages

Precision: the desired edit is encoded directed in nucleic acidsequence. Generality: in theory, could be possible to make any base pairconversion, including transversion edits, as well as small insertions ordeletions. There is a distinct editing window from that of base editorswith respect to Cas9 protospacer adjacent motif (PAM) sequence. Thismethod achieves many of the editing capabilities of homology-directedrepair (HDR), but without the major limitations of HDR (inefficient inmost cell types, and is usually accompanied by an excess of undesiredbyproducts such as indels). Also, it does not make double-stranded DNAbreaks (DSBs, so few indels, translocations, large deletions, p53activation, etc.

Example 2—Error-Prone Prime Editing (PE)

Prime editing (PE) systems described herein may also be used inconjunction with an error-prone reverse transcriptase enzyme to installmutations in a genome.

An embodiment is depicted in FIG. 22 , which is a schematic of anexemplary process for conducting targeted mutagenesis with anerror-prone reverse transcriptase on a target locus using a nucleic acidprogrammable DNA binding protein (napDNAbp) complexed with an extendedguide RNA. This process may be referred to as an embodiment of primeediting for targeted mutagenesis. The extended guide RNA comprises anextension at the 3′ or 5′ end of the guide RNA, or at an intramolecularlocation in the guide RNA. In step (a), the napDNAbp/gRNA complexcontacts the DNA molecule and the gRNA guides the napDNAbp to bind tothe target locus to be mutagenized. In step (b), a nick in one of thestrands of DNA of the target locus is introduced (e.g., by a nuclease orchemical agent), thereby creating an available 3′ end in one of thestrands of the target locus. In certain embodiments, the nick is createdin the strand of DNA that corresponds to the R-loop strand, i.e., thestrand that is not hybridized to the guide RNA sequence. In step (c),the 3′ end DNA strand interacts with the extended portion of the guideRNA in order to prime reverse transcription. In certain embodiments, the3′ ended DNA strand hybridizes to a specific RT priming sequence on theextended portion of the guide RNA. In step (d), an error-prone reversetranscriptase is introduced which synthesizes a mutagenized singlestrand of DNA from the 3′ end of the primed site towards the 3′ end ofthe guide RNA. Exemplary mutations are indicated with an asterisk “*”.This forms a single-strand DNA flap comprising the desired mutagenizedregion. In step (e), the napDNAbp and guide RNA are released. Steps (f)and (g) relate to the resolution of the single strand DNA flap(comprising the mutagenized region) such that the desired mutagenizedregion becomes incorporated into the target locus. This process can bedriven towards the desired product formation by removing thecorresponding 5′ endogenous DNA flap that forms once the 3′ singlestrand DNA flap invades and hybridizes to the complementary sequence onthe other strand. The process can also be driven towards productformation with second strand nicking, as exemplified in FIG. 1F.Following endogenous DNA repair and/or replication processes, themutagenized region becomes incorporated into both strands of DNA of theDNA locus.

Example 3—Trinucleotide Repeat Contraction with PE

The prime editing system or prime editing (PE) system described hereinmay be used to contract trinucleotide repeat mutations (or “tripletexpansion diseases”) to treating conditions such as Huntington's diseaseand other trinucleotide repeat disorders. Without wishing to be bound bytheory, triplet expansion is caused by slippage during DNA replicationor during DNA repair synthesis. Because the tandem repeats haveidentical sequence to one another, base pairing between two DNA strandscan take place at multiple points along the sequence. This may lead tothe formation of “loop out” structures during DNA replication or DNArepair synthesis. This may lead to repeated copying of the repeatedsequence, expanding the number of repeats. Additional mechanismsinvolving hybrid RNA:DNA intermediates have been proposed. Prime editingmay be used to reduce or eliminate these triplet expansion regions bydeletion one or more or the offending repeat codon triplets. In anembodiment of this use, FIG. 23 , provides a schematic of a PEgRNAdesign for contracting or reducing trinucleotide repeat sequences withprime editing.

Thus, prime editing may be able to be used to correct any trinucleotiderepeat disorder, including, Huntington's disease, Fragile X syndrome,and Friedreich's ataxia.

The most common trinucleotide repeat contains CAG triplets, though GAAtriplets (Friedreich's ataxia) and CGG triplets (Fragile X syndrome)also occur. The CAG triplets code for glutamine (Q), thus, CAG repeatsresult in polyglutamine tracts in the coding regions of diseasedproteins. This particular class of trinucleotide repeat disorders arealso called “polyglutamine (PolyQ) diseases.” Other trinucleotiderepeats can cause alterations in gene regulation and are referred to as“non-polyglutamine diseases.” Inheriting a predisposition to expansion,or acquiring an already expanded parental allele, increases thelikelihood of acquiring the disease. Pathogenic expansions oftrinucleotide repeats could be corrected using prime editing.

Prime editing may be implemented to contract triplet expansion regionsby nicking a region upstream of the triplet repeat region with the primeeditor comprising a PEgRNA appropriated targeted to the cut site. Theprime editor then synthesizes a new DNA strand (ssDNA flap) based on thePEgRNA as a template (i.e., the edit template thereof) that codes for ahealthy number of triplet repeats (which depends on the particular geneand disease). The newly synthesized ssDNA strand comprising the healthytriplet repeat sequence also is synthesized to include a short stretchof homology (i.e., the homology arm) that matches the sequence adjacentto the other end of the repeat. Invasion of the newly synthesizedstrand, and subsequent replacement of the endogenous DNA with the newlysynthesized ssDNA flap, leads to a contracted repeat allele.

Example 4—Peptide Tagging with PE

The prime editing systems (i.e., PE systems) described herein may alsobe used to introduce various peptide tags into protein coding genes.Such tags can include HEXA histidine tags, FLAG-tag, V5-tag, GCN4-tag,HA-tag, Myc-tag and others. This approach may be useful in applicationssuch as protein fluorescent labeling, immunoprecipitation,immunoblotting, immunohistochemistry, protein recruitment, inducibleprotein degrons, and genome-wide screening. Embodiments are depicted inFIGS. 25 and 26 .

FIG. 25 is a schematic showing gRNA design for peptide tagging genes atendogenous genomic loci and peptide tagging with TPRT genome editing(i.e., prime editing). The FlAsH and ReAsH tagging systems comprise twoparts: (1) a fluorophore-biarsenical probe, and (2) a geneticallyencoded peptide containing a tetracysteine motif, exemplified by thesequence FLNCCPGCCMEP (SEQ ID NO: 1). When expressed within cells,proteins containing the tetracysteine motif can be fluorescently labeledwith fluorophore-arsenic probes (see ref: J. Am. Chem. Soc., 2002, 124(21), pp 6063-6076. DOI: 10.1021/ja017687n). The “sortagging” systememploys bacterial sortase enzymes that covalently conjugate labeledpeptide probes to proteins containing suitable peptide substrates (seeref: Nat. Chem. Biol. 2007 November; 3(11):707-8. DOI:10.1038/nchembio.2007.31). The FLAG-tag (DYKDDDDK (SEQ ID NO: 2)),V5-tag (GKPIPNPLLGLDST (SEQ ID NO: 3)), GCN4-tag (EELLSKNYHLENEVARLKK(SEQ ID NO: 4)), HA-tag (YPYDVPDYA (SEQ ID NO: 5)), and Myc-tag(EQKLISEEDL (SEQ ID NO: 6)) are commonly employed as epitope tags forimmunoassays. The pi-clamp encodes a peptide sequence (FCPF) (SEQ ID NO:622) that can by labeled with a pentafluoro-aromatic substrates (ref:Nat. Chem. 2016 February; 8(2):120-8. doi: 10.1038/nchem.2413).

FIG. 26 shows precise installation of a His6-tag and a FLAG-tag intogenomic DNA. A guide RNA targeting the HEK3 locus was designed with areverse transcription template that encodes either an 18-nt His-taginsertion or a 24-nt FLAG-tag insertion. Editing efficiency intransfected HEK cells was assessed using amplicon sequencing. Note thatthe full 24-nt sequence of the FLAG-tag is outside of the viewing frame(sequencing confirmed full and precise insertion).

Example 5—Prevention or Treatment of Prion Disease with PE

This invention could help address the problem of prion disease inhumans, livestock, and wildlife. No previously described editingstrategy is efficient and clean enough to install protective mutationsor to reliably knock down PRNP. Cas9 nuclease and HDR can be used butwill generate mostly a mixture of PRNP indel variants some of which arethought to be pathogenic. Moreover, HDR does not work in most types ofcells. Prime editing is reliable and efficient at installing both typesof mutations without generating an excess of double-stranded DNA breaksor resulting indels.

This invention describes how to install a protective mutation in PRNPthat prevents or halts the progression of prion disease. This site isconserved in mammals, so in addition to treating human disease it couldalso be used to generate cows and sheep that are immune to priondisease, or even help cure wild populations of animals that aresuffering from prion disease. Prime editing has already been used toachieve ˜25% installation of a naturally occurring protective allele inhuman cells, and previous mouse experiments indicate that this level ofinstallation is sufficient to cause immunity from most prion diseases.This method is the first and potentially only current way to installthis allele with such high efficiency in most cell types. Anotherpossible strategy for treatment is to use prime editing to reduce oreliminate the expression of PRNP by installing an early stop codon inthe gene. Many researchers predict that doing so would treat thedisease.

Three potential therapeutic strategies include prime editing to reduceexpression of PrP. This goal may be accomplished by the introduction ofmutations that cause a premature stop codon in PRNP, eliminate the startcodon, mutate or delete essential amino acids codons, introduce orremove splice sites to generate an aberrant transcript, or alterregulatory elements that reduce transcript levels. Prime editing toeliminate disease mutations. Many variants of PRNP have been described(ncbi.nlm.nih.gov/pmc/articles/PMC6097508/#b154-ndt-14-2067) that leadto an increased likelihood of contracting disease. Each known variantcould be reversed using prime editing, since prime editing can make allpossible types of point mutations, local insertions, and localdeletions. Prime editing to introduce one or more protective mutationsinto PRNP that disrupt prion formation and/or transmission. For example,G127V in the human PRNP gene has been demonstrated to protect againstmany forms of prion disease (ncbi.nlm.nih.gov/pmc/articles/PMC4486072/).This mutation was later described to interfere with prion formation bypreventing formation of stable beta sheets and dimers(ncbi.nlm.nih.gov/pubmed/30181558, ncbi.nlm.nih.gov/pubmed/26906032). Inaddition to the introduction of single nucleotide polymorphisms, theinsertion or deletion of sequences in PRNP that would interfere withprion formation could also be used to protect from or treat priondisease.

The third therapeutic strategy is particularly advantageous because theintroduction of protective variants could confer a benefit even when arelatively small number of cells experience the edit. Furthermore, theintroduction of protective variants, especially those naturallyoccurring in human populations such as G127V, would not be expected tohave any detrimental consequences, while reducing expression of prionprotein as in strategy 1 could have some detrimental phenotypes, as havebeen documented in PRNP knock-out mice(ncbi.nlm.nih.gov/pmc/articles/PMC4601510/,ncbi.nlm.nih.gov/pmc/articles/PMC2634447/).

It has previously been demonstrated that mice expressing a ratio ofapproximately 2:1 of the wild type human prion protein: the protectiveG127V variant of the human prion protein (approximately 33% expressionof the protective variant) were entirely immune to most tested forms ofprion disease and were also resistant to variant Creutzfeldt-Jakobdisease (vCJD), the human disease transmitted from bovine spongiformencephalopathy (BSE, or mad cow disease)⁹¹. Mice that only expressed theprotective G127V variant were entirely immune to all tested priondisease challenges, including vCJD.

It is demonstrated herein that the protective G127V mutation can beefficiently installed in human cells in tissue culture using primeediting (see FIG. 27 ).

Informed by these results, three settings are described in which PRNPediting could be used. One setting PRNP editing can be used is primeediting in human patients to prevent or treat prion disease. A secondsetting PRNP editing can be used is prime editing in livestock toprevent the occurrence and spread of prion disease. Both cow and sheeplivestock have experienced sporadic occurrence of prion disease causedby the protein generated by the PRNP gene. In addition to thedebilitating and deadly disease suffered by the animal, these cases arealso economically devastating, in part due to the care that must betaken to prevent the spread of the extremely infectious disease. Asingle dairy cow in the state of Washington tested positive for BSE inDecember of 2003, which led to a projected loss of 2.8-4.2 billiondollars in beef sales the following yearbookstore.ksre.ksu.edu/pubs/MF2678.pdf). The PRNP gene is highlyconserved in mammals. Introducing a PRNP mutation such as G127V into thelivestock germline could eliminate the occurrence of BSE or scrapie, themanifestation of prion disease in sheep. A third setting PRNP editingcan be used is prime editing in wildlife could prevent the spread ofwild prion disease. Currently, cervid populations including deer, elk,and moose in North America are suffering from chronic wasting disease(CWD), a manifestation of prion disease caused by PNRP in these species.The occurrence has been reported to be as high as 25% in somepopulations (cdc.gov/prions/cwd/occurrence.html). CWD has also beenreported in Norway, Finland, and South Korea. It is not yet knownwhether the disease is transmissible from these species to humans(cdc.gov/prions/cwd/transmission.html) or livestock. The introduction ofPRNP mutations such as G127V in the germline of these species couldprotect them from CWD and reduce the risk of transmission to otherspecies including humans.

This method could be used to treat Creutzfeldt-Jakob Disease (CJD),kuru, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia(FFI), bovine spongiform encephalopathy (BSE; mad cow disease), scrapie(in sheep), and chronic wasting disease (CWD; in deer, elk, and moose).

The method would need to be combined with a delivery methodology forembryos or adult neurons, such as microinjection, lipid nanoparticles,or AAV vectors.

Example 6—RNA Tagging and Manipulation Using PE

A new method for the insertion of motifs into genetic sequences that tagor otherwise manipulate RNA within mammalian, eukaryotic, and bacterialcells is described herein. While it is estimated that only 1% of thehuman genome encodes proteins, virtually all of the genome istranscribed at some level. It is an open question how much of theresulting non-coding RNA (ncRNA) plays a functional role, let alone whatthe roles of most of these putative RNAs are. “Tagging” of these RNAmolecules via the insertion of a novel RNA-encoding sequence with auseful property into genes of interest is a useful method for studyingthe biological functions of RNA molecules in cells. It can also beuseful install tags onto protein-encoding mRNAs as a means to perturband thus better understand how mRNA modifications can affect cellularfunction. For instance, a ubiquitous natural RNA tag—polyadenylation—isused by cells to affect transport of mRNA into the cytoplasm. Differenttypes of polyadenylation signals result in different transport rates anddifferent mRNA lifespans and—thus—differences in the levels to which theencoded protein is expressed.

A common approach for expressing tagged RNAs within cells is toexogenously introduce a synthetic construct using either (i) transientplasmid transfection that produces a short-term burst of geneexpression, often at supraphysiologic levels; or (ii) permanentintegration of the tagged RNA gene into the genome (at random sites)using lentiviral integration or transposons, which enables prolongedexpression. Both of these approaches are limited by production ofaltered expression levels, and by the absence of natural mechanisms thatregulate the expression or activity of the gene. An alternative strategyis to directly tag a gene of interest at its endogenous locus usinghomology-directed repair (HDR) of double-stranded DNA breaks induced byCas9 or other targeted DNA nucleases. While this approach enables thegeneration of a wide range of endogenously tagged genes, HDR is markedlyinefficient and so requires significant screening to identify thedesired clonal population of cells that have been successfully tagged.Moreover, HDR is typically very inefficient or entirely inactive in alarge number of cell types, most notably in post-mitotic cells. The lowefficiency of HDR is further complicated by the generation of undesiredindel products, would could be especially problematic in the case of RNAgenes as they might lead to the production of an RNA whose activitiesinterfere with the function of normal alleles. Finally, researchersoften need to screen various tagging positions within an RNA molecule toachieve optimal performance. Combined, these drawbacks make HDR a lessdesirable method for installation of tags in RNA.

Prime editing is a new genome editing technology that enables targetedediting of genomic loci via the transfer of genetic information from RNAto DNA. Using prime editing, RNA genes could be tagged with a variety ofcomponents such as RNA aptamers, ribozymes, or other RNA motifs. Primeediting has the potential to be faster, cheaper, and effective in agreater variety of cell types by comparison to HDR strategies. As such,the described invention represents a novel, useful, and non-obvious toolfor investigating the biology of RNA genes in health and disease. A newmethod for the insertion of RNA motifs into genetic sequences that tagor otherwise manipulate RNA using prime editors (PEs) is describedherein. PEs are capable of site-specifically inserting, mutating, and/ordeleting multiple nucleotides at a desired genomic locus that istargetable by a CRISPR/Cas system. PEs are composed of fusions betweenCas9 nuclease domains and reverse transcriptase domains. They are guidedto their genomic target by engineered PEgRNAs (prime editing guideRNAs), which contain a guide spacer portion for DNA targeting, as wellas a template for reverse transcription that encodes the desired genomeedit (see FIG. 28A). It is envisioned that PE can be used to insertmotifs that are functional at the RNA level (hereafter RNA motifs) totag or otherwise manipulate non-coding RNAs or mRNAs. These motifs couldserve to increase gene expression, decrease gene expression, altersplicing, change post-transcriptional modification, affect thesub-cellular location of the RNA, enable isolation or determination ofthe intra- or extra-cellular location of the RNA (using, for instance,fluorescent RNA aptamers such as Spinach, Spinach2, Baby Spinach, orBroccoli), recruit endogenous or exogenous protein or RNA binders,introduce sgRNAs, or induce processing of the RNA, by eitherself-cleavage or RNAses (see FIG. 28B). Due to the flexibility of primeediting, it is not possible to provide a comprehensive list of RNAmotifs that could be installed within the genome. A series of examplesare shown here that broadly illustrate the predicted scope ofPE-installed RNA motifs that could be used to tag RNA genes. It iscurrently not possible to efficiently and fairly cleanly make thesechanges in most types of cells (including the many that do not supportHDR) using any other reported genome editing method besides PE.

Gene expression could be affected by encoding a 3′ untranslated region(UTR) that results in changes in nuclear transport or retention or mRNAlifespan. For instance, the polyA tail from polyomavirus simian virus 40(SV40) has additional helper sequences that enable efficienttranscription termination and can increase gene expression relative toother 3′ UTRs^(57,58). Example sequence of SV40 polyA tail:

SV40 POLYA TAIL (SEQ ID NO: 331)AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

Post-translational modification signals, besides polyadenylationsignals, could also be encoded by PE. These include signals incorporateN6-methyladenosine, N1-methyladenosine, 5-methylcytosine, andpseudouridine modifications⁵⁹. By using PE to include sequences bound byenzymes that write or remove these modifications within an RNAtranscript, it would be possible to induce their writing or erasing.This could be used as a tool to study the effects of these markers, toinduce cellular differentiation, affect stress responses, or, given thefunction of these markers are as yet underexplored, affect targetedcells in other fashions.

PE could encode mutations that affect subcellular localization. Forinstance, incorporation of tRNA-Lys within an mRNA can theoreticallyresult in transport to the mitochondria⁶⁰, while various 3′ UTRs canresult in nuclear retention or transport⁶¹.

Examples

SV40 polyA signal results in transport.

SV40 POLYA TAIL (SEQ ID NO: 331)AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

U1 snRNA 3′ box results in retention.

U1 SNRNA 3′ BOX (SEQ ID NO: 625)TTCATTCAGCAAGTTCAGAGAAATCTGAACTTGCTGGATTTTTGGAGCAGGGAGATGGAATAGGAGCTTGCTCCGTCCACTCCACGCATCGACCTGGTATTGCAGTACCTCCAGGAACGGTGCACCCACTTTCTGGAGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTT GGCGTCTTAA

Determining the sub-cellular localization of endogenous RNA can bechallenging and requires the addition of exogenous, fluorescently-taggednucleotide probes, as in the case of FISH, or time-consuming andpotentially inaccurate cell fractionation followed by RNA detection.Encoding a probe within the endogenous RNA would obviate many of theseissues. One example would be to encode a fluorescent RNA aptamer, suchas Spinach⁶² or Broccoli within an endogenous RNA, thereby visualizingthe presence of that RNA via addition of a small moleculeproto-fluorophore.

Broccoli Aptamer:

BROCCOLI APTAMER (SEQ ID NO: 357)GAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTC

PE could insert or remove sequences that encode RNA that are recognizedby RNA binding proteins, affecting RNA stability, expression,localization, or modification (for instance, see proteins listed⁶³)

PE could insert sequences that encode sgRNAs within the genome, as aviral or cancer defense mechanism. Similarly, it could be used to insertmicroRNAs (e.g., pre-microRNAs) to direct silencing of targeted genes.

PE could insert sequences resulting in processing of the RNA, either byitself, or by external factors, either as a therapy or tool for studyingthe function of various portions of the RNA. For instance, the HDVribozyme⁶⁴, when included within an RNA sequence, results in processingof the RNA immediately 5′ of the ribozyme, while the hammerhead ribozymecleaves prior to the third stem within the ribozyme⁶⁵. Otherself-cleaving ribozymes include pistol⁶⁶, hatchet⁶⁶, hairpin⁶⁷,Neuropora Varkud satellite⁶⁸, glmS⁶⁹, twister⁷⁰, and twister sister⁶⁶.These sequences could include wild-type or engineered or evolvedversions of ribozymes. The majority of these ribozymes could havedifferent sequences depending on the region of RNA into which they wereassociated, depending on where the ribozyme cut site is located.Sequences that would direct the processing of the RNA by externalfactors, such as sequence specific RNAses⁷¹, RNAses that recognizespecific structures⁷²—such as Dicer⁷³ and Drosha⁷⁴, could also beachieved.

HDV Ribozyme:

HDV RIBOZYME (SEQ ID NO: 365)GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTT CGGCATGGCGAATGGGAC

REFERENCES FOR EXAMPLE 6

The following references are incorporated herein by reference in theirentireties.

-   1. Schek N, Cooke C, Alwine J C. Molecular and Cellular Biology.    1992.-   2. Gil A, Proudfoot N J. Cell. 1987.-   3. Zhao, B. S., Roundtree, I. A., He, C. Nat Rev Mol Cell Biol.    2017.-   4. Rubio, M. A. T., Hopper, A. K. Wiley Interdiscip Rev RNA 2011.-   5. Shechner, D. M., Hacisuleyman E., Younger, S. T., Rinn, J. L. Nat    Methods. 2015.-   6. Paige, J. S., Wu, K. Y., Jaffrey, S. R. Science 2011.-   7. Ray D., . . . Hughes T R. Nature 2013.-   8. Chadalavada, D. M., Cerrone-Szakal, A. L., Bevilacqua, P. C. RNA    2007.-   9. Forster A C, Symons R H. Cell. 1987.-   10. Weinberg Z, Kim P B, Chen T H, Li S, Harris K A, Lünse C E,    Breaker R R. Nat. Chem. Biol. 2015.-   11. Feldstein P A, Buzayan J M, Bruening G. Gene 1989.-   12. Saville B J, Collins R A. Cell. 1990.-   13. Winkler W C, Nahvi A, Roth A, Collins J A, Breaker R R. Nature    2004.-   14. Roth A, Weinberg Z, Chen A G, Kim P G, Ames T D, Breaker R R.    Nat Chem Biol. 2013.-   15. Choudhury R, Tsai Y S, Dominguez D, Wang Y, Wang Z. Nat Commun.    2012.-   16. MacRae I, Doudna J A. Curr Opin Struct Biol. 2007.-   17. Bernstein E, Caudy A A, Hammond S M, Hannon G J Nature 2001.-   18. Filippov V, Solovyev V, Filippova M, Gill S S. Gene 2000.

Example 7—Generation of Gene Libraries with PE

A new method for the cellular generation of highly sophisticatedlibraries of protein- or RNA-coding genes with defined or variableinsertions, deletions, or defined amino acid/nucleotide conversions, andtheir use in high-throughput screening and directed evolution isdescribed herein. The references cited in the Example are based from thelist of references included at the end of this Example.

The generation of variable genetic libraries has most commonly beenaccomplished through mutagenic PCR¹. This method relies on either usingreaction conditions that reduce the fidelity of DNA polymerase, or usingmodified DNA polymerases with higher mutation rates. As such, biases inthese polymerases are reflected in the library product (e.g. apreference for transition mutations versus transversions). An inherentlimitation of this approach to library construction is a relativeinability to affect the size of the gene being varied. Most DNApolymerases have extremely low rates of indel mutations² (insertions ordeletions), and most of these will result in frameshift mutations inprotein-coding regions, rendering members of the library unlikely topass any downstream selection. Additionally, biases in PCR and cloningcan make it difficult to generate single libraries consisting of genesof different sizes. These limitations can severely limit the efficacy ofdirected evolution to enhance existing or engineer novel proteinfunctions. In natural evolution, large changes in protein function orefficacy are typically associated with insertion and deletion mutationsthat are unlikely to occur during canonical library generation formutagenesis. Furthermore, these mutations most commonly occur in regionsof the protein in question that are predicted to form loops, as opposedto the hydrophobic core. Thus, most indels generated using a traditionalunbiased approach are likely to either be deleterious or ineffective.

Libraries that could bias such mutations to the sites within the proteinwhere they would be most likely to be beneficial, i.e. loop regions,would have a significant advantage over traditional libraries given thatall libraries access only a fraction of the possible mutation space.Finally, although it is possible to generate genetic libraries withsite-specific indel mutations through multistep PCR and clonal assemblyusing NNK primers or via DNA shuffling, these libraries cannot undergoadditional rounds of ‘indelgenesis’ in continual evolution. Continuousevolution is a type of directed evolution with minimal userintervention. One such example is PACE³. Because continuous evolutionoccurs with minimal user intervention, any increase in library diversityduring the evolution must occur using the native replication machinery.As such, although libraries of genes with inserted or removed codons asspecific loci can be generated and screened in PACE, additional roundsof ‘indelgenesis’ are not possible.

It is envisioned that the programmability of prime editing (PE) can beleveraged to generate highly sophisticated, programmed genetic librariesfor use in high-throughput screening and directed evolution (see FIG.29A). PE can insert, change or remove defined numbers of nucleotidesfrom specified genetic loci using information encoded in a prime editingguide RNA (PEgRNA) (see FIG. 29B). This enables the generation oftargeted libraries with one or more amino acids inserted or removed fromthe loop regions wherein mutations are most likely to give rise tochanges in function, without background introduction of nonfunctionalframeshift mutations (see FIG. 29C). PE can be used to install specificsets of mutations without regard for biases inherent in either DNApolymerase or the sequence being mutated.

For instance, while converting a CCC codon to a stop codon would be anunlikely occurrence via canonical library generation because it wouldrequire three consecutive mutations, including two transversions, PEcould be used to convert any given, targeted codon to a TGA stop codonin one step. They could also be used to install programmed diversity atgiven positions, for instance by incorporating codons encoding anyhydrophobic amino acid at a given site, while not encoding any others.Furthermore, because of the programmability of PE, multiple PEgRNAscould be utilized to generate multiple different edits at multiple sitessimultaneously, enabling the generation of highly programmed libraries(see FIG. 29D). Additionally, it is possible to use reversetranscriptases with lower fidelity to generate regions of mutagenesiswithin an otherwise invariable library (such as the HIV-I reversetranscriptase⁴ or Bordetella phage reverse transcriptase⁵).

The possibility of iterative rounds of PE on the same site is alsoenvisioned, allowing—for instance—the repeated insertion of codons at asingle site. Finally, it is envisioned that all of the above describedapproaches can be incorporated into continual evolution, enabling thegeneration of novel in situ evolving libraries (see FIG. 30 ). Theycould also be used to construct these libraries within other cell typeswhere it would otherwise be difficult to assemble large libraries, forinstance within mammalian cells. Generation of PE-encoding bacterialstrains that have been optimized for directed evolution would be auseful additional tool for the identification of proteins and RNAs withimproved or novel functionality. All of these uses of PE are non-obviousdue to the novel nature of PEs. In conclusion, library generation via PEwould be a highly useful tool in synthetic biology and directedevolution, as well as for high-throughput screening of protein and RNAcombinatorial mutants.

Competing Approaches

The chief method by which diverse libraries are currently generated isby mutagenic PCR¹, described above. Insertions or deletions can beintroduced via degenerated NNK primers at defined sites during PCR,although introducing such mutations at multiple sites requires multiplerounds of iterative PCR and cloning before constructing a more diverselibrary via mutagenic PCR, rendering the method slow. An alternative,complementary method is DNA shuffling, where fragments of a library ofgenes generated via DNase treatment are introduced into a PCR reactionwithout primers, resulting in the annealing of different fragments toeach other and the rapid generation of more diverse libraries than viamutagenic PCR alone⁶. Although this approach can theoretically generateindel mutations, it more often results in frameshift mutations thatdestroy gene function. Furthermore, DNA shuffling requires a high degreeof homology between gene fragments. Both of these methods must be donein vitro, with the resulting library transformed into cells, whilelibraries generated by PE can be constructed in situ, enabling their usein continual evolution. While libraries can be constructed in situthrough in vivo mutagenesis, these libraries rely on the host cellularmachinery and exhibit biases against indels. Similarly, althoughtraditional cloning methods can be used to generate site-specificmutational profiles, they cannot be used in situ and are generallyassembled one at a time in vitro before being transformed into cells.The efficiency and broad functionality of PE in both prokaryotic andeukaryotic cell types further suggests that these libraries could beconstructed directly in the cell type of interest, as opposed to beingcloned into a model organism such as E. coli and then transferred intothe cell or organism of interest. Another competing approach fortargeted diversification is automated multiplex genome engineering, orMAGE, wherein multiple single-stranded DNA oligonucleotides can beincorporated within replication forks and result in programmablemutations⁷. However, MAGE requires significant modification of the hoststrain and can lead to a 100-fold increase in off-target or backgroundmutations⁸, whereas PE is more highly programmed and anticipated toresult in fewer off-target effects. Additionally, MAGE has not beendemonstrated in a wide variety of cell types, including mammalian cells.Prime editing is a novel and non-obvious complementary technique forlibrary generation.

Examples of PE in Directed Evolution to Construct Gene Libraries

In one example, PE can be used in a directed evolution experiment tointroduce protein variants into gene libraries during a continualevolution experiment using PACE, permitting iterative accumulation ofboth point mutations and indels in a manner not possible via traditionalapproaches. It has already been shown that PE can site-specifically andprogrammably insert nucleotides into a genetic sequence in E. coli. Inthe outlined directed evolution, it is proposed to identify monobodieswith improved binding to a specific epitope via a modified two-hybridprotein:protein binding PACE selection. Specific and highly variableloops within these monobodies contribute significantly to affinity andspecificity. Improved monobody binding might be obtained rapidly in PACEby varying the length and composition of these loops in a targetedfashion. However, varying sequence length is not an establishedfunctionality of PACE. While library of varied loop sizes might be usedas a starting point for PACE, no subsequent improvements to length wouldarise throughout the PACE selection, barring access to beneficialsynergistic combinations of point mutations and indel mutations.Introducing PE to the PACE selection would enable the in situ generationand evolution of monobodies with varying loop lengths. To do so, it isenvisioned the introduction of an additional PE plasmid to the host E.coli strain, encoding the PE enzyme and one or more PEgRNAs. Expressionof PE enzyme and PEgRNAs would be under the control of a small moleculedelivered to the PACE lagoon at a rate selected by the experimenter.

In various embodiments, the PEgRNA components would contain a spacerdirecting the PE to the site of interest on the selection phage andwould be designed such that a multiple of three nucleotides could beinserted at the target site such that a new PEgRNA binding site would beintroduced, enabling the iterative insertion of one or more codons atthe targeted site.

In parallel, another host E. coli strain might include PEgRNAs thatwould template the removal of one or more codons, enabling loop size toshrink during the evolution. A PACE experiment might utilize a mixtureof both strains or alternate the two to permit the slow and controlledaddition or removal of loop sequences.

It is noted that this technique can also be applied to the evolution ofantibodies. The binding principles governing antibodies are very similarto those governing monobodies: the length of antibodycomplement-determining region loops is critical to their bindingfunction. Further, longer loop lengths have been found to be critical inthe development of rare antibodies with broadly protective activityagainst HIV-1 and other viral infections⁹. Application of PE asdescribed above to an antibody or antibody-derived molecule would permitthe generation of antibodies with diverse loop length and varied loopsequence. In combination with PACE, such an approach would permitenhanced binding through loop geometries not accessible to standardPACE, and thus permit evolution of highly functional antibodies.

Experiments will show the ability to use PE to correct a deleteriousmutation in bacteriophage M3 in phage-assisted non-continuous evolution(PANCE), a necessary first step for using PE in continuous evolution(see FIG. 69 ).

REFERENCES FOR EXAMPLE 7

The following references are incorporated herein by reference in theirentireties.

-   1. Cadwell R C and Joyce G F. PCR Methods Appl. 1992.-   2. McInerney P, Adams P, and Hadi M Z. Mol Biol Int. 2014.-   3. Esvelt K M, Carlson J C, and Liu D R. Nature. 2011.-   4. Naorem S S, Hin J, Wang S, Lee W R, Heng X, Miller J F, Guo H.    Proc Natl Acad Sci USA 2017.-   5. Martinez M A, Vartanian J P, Wain-Hobson S. Proc Natl Acad Sci    USA 1994.-   6. Meyer A J, Ellefson J W, Ellington A D. Curr Protoc Mol Biol.    2014.-   7. Wang H H, Isaacs F J, Carr P A, Sun Z Z, Xu G, Forest C R, Church    G M. Nature. 2009.-   8. Nyerges Á et al. Proc Natl Acad Sci USA. 2016.-   9. Mascola J R, Haynes B F. Immunol Rev. 2013.

Example 8—Immunoepitope Insertion by PE

Precise genome targeting technologies using the CRISPR/Cas system haverecently been explored in a wide range of applications, including theinsertion of engineered DNA sequences into targeted genomic loci.Previously, homology-directed repair (HDR) has been used for thisapplication, requiring an ssDNA donor template and repair initiation bymeans of a double-stranded DNA break (DSB). This strategy offers thebroadest range of possible changes to be made in cells and is the onlymethod available to insert large DNA sequences into mammalian cells.However, HDR is hampered by undesired cellular side effects stemmingfrom its initiating DSB, such as high levels of indel formation, DNAtranslocations, large deletions, and P53 activation. In addition tothese drawbacks, HDR is limited by low efficiency in many cell types (Tcells are a notable exception to this observation). Recent efforts toovercome these drawbacks include fusing human Rad51 mutants to a Cas9D10A nickase (RDN), resulting in a DSB-free HDR system that featuresimproved HDR product:indel ratios and lower off target editing, but isstill hampered by cell-type dependencies and only modest HDR editingefficiency.

Recently developed fusions of Cas9 to reverse transcriptases (“Primeeditors”) coupled with PEgRNAs represent a novel genome editingtechnology that offers a number of advantages over existing genomeediting methods, including the ability to install any single nucleotidesubstitution, and to insert or delete any short stretch of nucleotides(up to at least several dozen bases) in a site-specific manner. Notably,PE edits are achieved with generally low rates of unintended indels. Assuch, PE enables targeted insertion-based editing applications that havebeen previously impossible or impractical.

This particular invention describes a method for using prime editing asa means to insert known immunogenicity epitopes into endogenous orforeign genomic DNA, resulting in modification of the correspondingproteins for therapeutic or biotechnological applications (see FIGS. 31and 32 ). Prior to the invention of prime editing, such insertions couldbe achieved only inefficiently and with high rates of indel formationfrom DSBs. prime editing solves the problem of high indel formation frominsertion edits while generally offering higher efficiency than HDR.This lower rate of indel formation presents a major advantage of primeediting over HDR as a method for targeted DNA insertions, especially inthe described application of inserting immunogenicity epitopes. Thelength of epitopes is in a range from few bases to hundreds of bases.Prime editor is the most efficient and cleanest technology to achievesuch targeted insertions in mammalian cells.

The key concept of the invention is the use of prime editors to insert anucleotide sequence containing previously described immunogenicityepitopes into endogenous or foreign genomic DNA for the downregulationand/or destruction of their protein products and/or expressing celltypes. Nucleotide sequences for immunogenic epitope insertion would betargeted to genes in a manner to produce fusion proteins of the targetedgene's coded protein and the inserted immunogenic epitope'scorresponding protein translation. Patient's immune systems will havebeen previously trained to recognize these epitopes as a result ofstandard prior immunization from routine vaccination against, forexample, tetanus or diphtheria or measles. As a result of theimmunogenic nature of the fused epitopes, patient's immune systems wouldbe expected to recognize and disable the prime edited protein (not justthe inserted epitope) and potentially the cells from which it wasexpressed.

Fusions to targeted genes would be engineered as needed to ensure theinserted epitope protein translation is exposed for immune systemrecognition. This could include targeted nucleotide insertions resultingin protein translations yielding C-terminal fusions of immunogenicityepitopes to targeted genes, N-terminal fusions of immunogenicityepitopes to targeted genes, or the insertion of nucleotides into genesso that immunogenicity epitopes are coded within surfaced-exposedregions of protein structure.

Protein linkers encoded as nucleotides inserted between the target genesequence and the inserted immunogenicity epitope nucleotide sequence mayneed to be engineered as part of this invention to facilitate immunesystem recognition, cellular trafficking, protein function, or proteinfolding of the targeted gene. These inserted nucleotide-encoded proteinlinkers may include (but are not limited to) variable lengths andsequences of the XTEN linker or variable lengths and sequences ofGlycine-Serine linkers. These engineered linkers have been previous usedto successfully facilitate protein fusions.

Distinguishing features of this invention include the ability to usepreviously acquired immune responses to specific amino acid sequences asa means to induce an immune response against otherwise non-immunogenicproteins. Another distinguishing feature is the ability to insert thenucleotide sequences of these immunogenic epitopes in a targets mannerthat does not induce high levels of unwanted indels as a by-productediting and is efficient in its insertion. The invention discussedherein also has the ability to combine cell type-specific deliverymethods (such as AAV serotypes) to insert epitopes in cell types thatare of interest to trigger an immune response to.

Prime editing as a means of inserting immunogenic epitopes intopathogenic genes could be used to program the patient's immune system tofight a wide variety of diseases (not limited to cancer as withimmuno-oncology strategies). An immediately relevant use of thistechnology would be as a cancer therapeutic as it could undermine atumor's immune escape mechanism by causing an immune response to arelevant oncogene like HER2 or growth factors like EGFR. Such anapproach could seem similar to T-cell engineering, but one novel advanceof this approach is that it can be utilized in many cell types and fordiseases beyond cancer, without needed to generate and introduceengineered T-cells into patients.

Using PE to insert an immunogenicity epitope which most people arealready vaccinated against (tetanus, pertussis, diphtheria, measles,mumps, rubella, etc.) into a foreign or endogenous gene that drives adisease, so the patient's immune system learns to disable that protein.

Diseases that stand to have a potential therapeutic benefit from theaforementioned strategy include those caused by aggregation of toxicproteins, such as in fatal familial insomnia. Other diseases that couldbenefit include those caused by pathogenic overexpression of anotherwise nontoxic endogenous protein, and those caused by foreignpathogens.

Primary therapeutic indications include those mentioned above such astherapeutics for cancer, prion and other neurodegenerative diseases,infectious diseases, and preventative medicine. Secondary therapeuticindications may include preventative care for patients with late-onsetgenetic diseases. It is expected that current standard of care medicinesmay be used in conjunction with prime editing for some diseases, likeparticularly aggressive cancers, or in cases where medications helpalleviate disease symptoms until the disease completely cured.

Below are examples of immunogenic epitopes that can by inserted by primeediting can be used to achieve:

Epitope Amino Acid Vaccine Disease SequenceExample Nucleic Acid Sequence (8) 1 Tetanus QYIKANSKFIGITECATGATATAAAAGCAAATTCTAAAT toxoid L (SEQ ID NO: 396)TTATAGGTATAACTGAACTA (SEQ ID NO: 397) 2 Diphtheria GADDVVDSSKSFGGCGCCGACGACGTGGTGGACAGCA toxin mutant VMENFSSYHGTKGCAAGAGCTTCGTGATGGAGAACTT CRM197 PGYVDSIQKGIQK CAGCAGCTACCACGGCACCAAGCCCPKSGTQGNYDDD GGCTACGTGGACAGCATCCAGAAGG WKEFYSTDNKYDGCATCCAGAAGCCCAAGAGCGGCAC AAGYSVDNENPL CCAGGGCAACTACGACGACGACTGGSGKAGGVVKVTY AAGGAGTTCTACAGCACCGACAACA PGLTKVLALKVDAGTACGACGCCGCCGGCTACAGCGT NAETIKKELGLSL GGACAACGAGAACCCCCTGAGCGGCTEPLMEQVGTEEF AAGGCCGGCGGCGTGGTGAAGGTGA IKRFGDGASRVVLCCTACCCCGGCCTGACCAAGGTGCT SLPFAEGSSSVEYI GGCCCTGAAGGTGGACAACGCCGAGNNWEQAKALSVE ACCATCAAGAAGGAGCTGGGCCTGA LEINFETRGKRGQGCCTGACCGAGCCCCTGATGGAGCA DAMYEYMAQAC GGTGGGCACCGAGGAGTTCATCAAGAGNRVRRSVGSS AGGTTCGGCGACGGCGCCAGCAGGG LSCINLDWDVIRDTGGTGCTGAGCCTGCCCTTCGCCGA KTKTKIESLKEHG GGGCAGCAGCAGCGTGGAGTACATCPIKNKMSESPNKT AACAACTGGGAGCAGGCCAAGGCCC VSEEKAKQYLEEFTGAGCGTGGAGCTGGAGATCAACTT HQTALEHPELSEL CGAGACCAGGGGCAAGAGGGGCCAKTVTGTNPVFAG GGACGCCATGTACGAGTACATGGCC ANYAAWAVNVACAGGCCTGCGCCGGCAACAGGGTGA QVIDSETADNLEK GGAGGAGCGTGGGCAGCAGCCTGATTAALSILPGIGSV GCTGCATCAACCTGGACTGGGACGT MGIADGAVHHNTGATCAGGGACAAGACCAAGACCAA EEIVAQSIALSSL GATCGAGAGCCTGAAGGAGCACGGCMVAQAIPLVGEL CCCATCAAGAACAAGATGAGCGAGA VDIGFAAYNFVESGCCCCAACAAGACCGTGAGCGAGGA IINLFQVVHNSYN GAAGGCCAAGCAGTACCTGGAGGARPAYSPGHKTQPF GTTCCACCAGACCGCCCTGGAGCAC LHDGYAVSWNTVCCCGAGCTGAGCGAGCTGAAGACCG EDSIIRTGFQGESG TGACCGGCACCAACCCCGTGTTCGCHDIKITAENTPLPI CGGCGCCAACTACGCCGCCTGGGCC AGVLLPTIPGKLDGTGAACGTGGCCCAGGTGATCGACA VNKSKTHISVNGR GCGAGACCGCCGACAACCTGGAGAAKIRMRCRAIDGD GACCACCGCCGCCCTGAGCATCCTG VTFCRPKSPVYVGCCCGGCATCGGCAGCGTGATGGGCA NGVHANLHVAFH TCGCCGACGGCGCCGTGCACCACAARSSSEKIHSNEISS CACCGAGGAGATCGTGGCCCAGAGC DSIGVLGYQKTVATCGCCCTGAGCAGCCTGATGGTGG DHTKVNSKLSLFF CCCAGGCCATCCCCCTGGTGGGCGAEIKS (SEQ ID NO: GCTGGTGGACATCGGCTTCGCCGCC 630)TACAACTTCGTGGAGAGCATCATCA ACCTGTTCCAGGTGGTGCACAACAGCTACAACAGGCCCGCCTACAGCCCC GGCCACAAGACCCAGCCCTTCCTGCACGACGGCTACGCCGTGAGCTGGAA CACCGTGGAGGACAGCATCATCAGGACCGGCTTCCAGGGCGAGAGCGGCC ACGACATCAAGATCACCGCCGAGAACACCCCCCTGCCCATCGCCGGCGTG CTGCTGCCCACCATCCCCGGCAAGCTGGACGTGAACAAGAGCAAGACCCA CATCAGCGTGAACGGCAGGAAGATCAGGATGAGGTGCAGGGCCATCGACG GCGACGTGACCTTCTGCAGGCCCAAGAGCCCCGTGTACGTGGGCAACGGC GTGCACGCCAACCTGCACGTGGCCTTCCACAGGAGCAGCAGCGAGAAGAT CCACAGCAACGAGATCAGCAGCGACAGCATCGGCGTGCTGGGCTACCAGA AGACCGTGGACCACACCAAGGTGAACAGCAAGCTGAGCCTGTTCTTCGAG ATCAAGAGC (SEQ ID NO: 399) 3 mumpsGTYRLIPNARANL GGCACCTACAGGCTGATCCCCAACG TA (SEQ ID NO:CCAGGGCCAACCTGACCGCC (SEQ 400) ID NO: 401) 4 mumps PSKFFTISDSATFACcgagcaaattttttaccattagcgatagcgcgacctttgcg PGPVSNA (SEQ IDccgggcccggtgagcaacgcg NO: 402) (SEQ ID NO: 403) PSKLFIMLDNATFCcgagcaaactgtttattatgctggataacgcgacctttgcg APGPVVNA (SEQccgggcccggtggtgaacgcg ID NO: 404) (SEQ ID NO: 405)Selected examples from Hemagglutinin- neuraminidase (HN) diversity amongoutbreak strains (table1) Divergencebetween vaccine strain JL5 and outbreak strains (tab1e2) 5 Rubella virusTPPPYQVSCGGES ACCCCCCCCCCCTACCAGGTGAGCT (RV) DRASARVIDPAAGCGGCGGCGAGAGCGACAGGGCCA QS (SEQ ID NO: GCGCCAGGGTGATCGACCCCGCCGC 406)CCAGAGC (SEQ ID NO: 407) 6 Hemagglutinin PEYAYKIVKNKKCCCGAGTACGCCTACAAGATCGTGA MEDGFLQGMVD AGAACAAGAAGATGGAGGACGGCTGWYGHHSNEQGS TCCTGCAGGGCATGGTGGACGGCTG GLMENERTLDKAGTACGGCCACCACAGCAACGAGCAG NPNNDLCSWSDH GGCAGCGGCCTGATGGAGAACGAGEASSNNTNQEDLL AGGACCCTGGACAAGGCCAACCCCA QRESRRKKRIGTSACAACGACCTGTGCAGCTGGAGCGA TLNQRGNCNTKC CCACGAGGCCAGCAGCAACAACACCQTEEARLKREEVS AACCAGGAGGACCTGCTGCAGAGGG LVKSDQCSNGSLAGAGCAGGAGGAAGAAGAGGATCG QCRANNSTEQVD GCACCAGCACCCTGAACCAGAGGGG(SEQ ID NO: 408) CAACTGCAACACCAAGTGCCAGACC GAGGAGGCCAGGCTGAAGAGGGAGGAGGTGAGCCTGGTGAAGAGCGACC AGTGCAGCAACGGCAGCCTGCAGTGCAGGGCCAACAACAGCACCGAGCA GGTGGAC (SEQ ID NO: 409) 7 NeuraminidaseTKSTNSRSGGISG ACCAAGAGCACCAACAGCAGGAGC PDNEAPVGEAPSPGGCGGCATCAGCGGCCCCGACAACG YGDNPRPNDGNN AGGCCCCCGTGGGCGAGGCCCCCAGIRIGSKGYNGIITD CCCCTACGGCGACAACCCCAGGCCC TIEESCSCYPDAKAACGACGGCAACAACATCAGGATCG VVKSVELDSTIWT GCAGCAAGGGCTACAACGGCATCATSGSSPNQKIITIGW CACCGACACCATCGAGGAGAGCTGC DPNGWTGTPMSPAGCTGCTACCCCGACGCCAAGGTGG NGAYGTDGPSNG TGAAGAGCGTGGAGCTGGACAGCACQANQHQAESISA CATCTGGACCAGCGGCAGCAGCCCC GNSSLCPIRDNWHAACCAGAAGATCATCACCATCGGCT GSNRSWSWPDGA GGGACCCCAACGGCTGGACCGGCACE (SEQ ID NO: 410) CCCCATGAGCCCCAACGGCGCCTAC GGCACCGACGGCCCCAGCAACGGCCAGGCCAACCAGCACCAGGCCGAGA GCATCAGCGCCGGCAACAGCAGCCTGTGCCCCATCAGGGACAACTGGCAC GGCAGCAACAGGAGCTGGAGCTGGCCCGACGGCGCCGAG (SEQ ID NO: 411) 8 TAP EKIVLLLAMMEKIGAGAAGATCGTGCTGCTGCTGGCCA (transport VLLLAKCQTPMGTGATGGAGAAGATCGTGCTGCTGCT antigen AIKAVDGVTNKC GGCCAAGTGCCAGACCCCCATGGGCpresentation) PYLGSPSF (SEQ GCCATCAAGGCCGTGGACGGCGTGA on H5N1ID NO: 412) CCAACAAGTGCCCCTACCTGGGCAG virus CCCCAGCTTC (SEQ ID NO: 413)hemagglutinin 9 TAP IRPCFWVELNPNQ ATCAGGCCCTGCTTCTGGGTGGAGC (transportKIITIRPCFWVELI TGAACCCCAACCAGAAGATCATCAC antigen CYPDAGEIT (SEQCATCAGGCCCTGCTTCTGGGTGGAG presentation) ID NO: 414)CTGATCTGCTACCCCGACGCCGGCG on h5n1 virus AGATCACC (SEQ ID NO: 415)neuraminidase 10 hemagglutinin MEKIVLLLAEKIV ATGGAGAAGATCGTGCTGCTGCTGGepitopes LLLAMCPYLGSPS CCGAGAAGATCGTGCTGCTGCTGGC toward class IFKCQTPMGAIKA CATGTGCCCCTACCTGGGCAGCCCC HLA VDGVTNK (SEQAGCTTCAAGTGCCAGACCCCCATGG ID NO: 416) GCGCCATCAAGGCCGTGGACGGCGTGACCAACAAG (SEQ ID NO: 417) 11 neuraminidase NPNQKIITICYPDAGAACCCCAACCAGAAGATCATCACCAT epitopes EITIRPCFWVELRPCCTGCTACCCCGACGCCGGCGAGATCA toward class I FWVELI (SEQ IDCCATCAGGCCCTGCTTCTGGGTGGAG HLA NO: 418) CTGAGGCCCTGCTTCTGGGTGGAGCTGATC (SEQ ID NO: 419) 12 hemagglutinin MVSLVKSDQIGTSTATGGTGAGCCTGGTGAAGAGCGACCA epitopes LNQR (SEQ ID NO:GATCGGCACCAGCACCCTGAACCAGA toward class II 420) GG (SEQ ID NO: 421) HLA13 neuraminidase YNGIITDTI (SEQ ID TACAACGGCATCATCACCGACACCAT epitopesNO: 422) C (SEQ ID NO: 423) toward class II HLA 14 hemagglutininMEKIVLLLAEKIVL ATGGAGAAGATCGTGCTGCTGCTGGC epitope LLAMMVSLVKSDQCGAGAAGATCGTGCTGCTGCTGGCCA H5N1-bound CPYLGSPSFIGTSTLTGATGGTGAGCCTGGTGAAGAGCGAC class I and NQRKCQTPMGAIKCAGTGCCCCTACCTGGGCAGCCCCAG class II HLA AVDGVTNK (SEQCTTCATCGGCACCAGCACCCTGAACC ID NO: 424) AGAGG (SEQ ID NO: 425) 15neuraminidase NPNQKIITIYNGIIT AACCCCAACCAGAAGATCATCACCAT epitopeDTICYPDAGEITIRP CTACAACGGCATCATCACCGACACCA H5N1-bound CFWVELRPCFWVETCTGCTACCCCGACGCCGGCGAGATC class I and LI (SEQ ID NO: 426)ACCATCAGGCCCTGCTTCTGGGTGGA class II HLA GCTGAGGCCCTGCTTCTGGGTGGAGCTGATC (SEQ ID NO: 427)

Below are additional examples of epitopes that may be integrated into atarget gene for immunoepitope tagging:

REFERENCES CITED IN EXAMPLE 8

The following references are incorporated by reference in theirentireties.

-   1. X. Wen, K. Wen, D. Cao, G. Li, R. W. Jones, J. Li, S. Szu, Y.    Hoshino, L. Yuan, Inclusion of a universal tetanus toxoid CD4(+) T    cell epitope P2 significantly enhanced the immunogenicity of    recombinant rotavirus ΔVP8* subunit parenteral vaccines. Vaccine 32,    4420-4427 (2014).-   2. G. Ada, D. Isaacs, Carbohydrate-protein conjugate vaccines. Clin    Microbiol Infect 9, 79-85 (2003).-   3. E. Malito, B. Bursulaya, C. Chen, P. L. Surdo, M. Picchianti, E.    Balducci, M. Biancucci, A. Brock, F. Berti, M. J. Bottomley, M.    Nissum, P. Costantino, R. Rappuoli, G. Spraggon, Structural basis    for lack of toxicity of the diphtheria toxin mutant CRM197.    Proceedings of the National Academy of Sciences 109, 5229 (2012).-   4. J. de Wit, M. E. Emmelot, M. C. M. Poelen, J.    Lanfermeijer, W. G. H. Han, C. van Els, P. Kaaijk, The Human CD4(+)    T Cell Response against Mumps Virus Targets a Broadly Recognized    Nucleoprotein Epitope. J Virol 93, (2019).-   5. M. May, C. A. Rieder, R. J. Rowe, Emergent lineages of mumps    virus suggest the need for a polyvalent vaccine. Int J Infect Dis    66, 1-4 (2018).-   6. M. Ramamurthy, P. Rajendiran, N. Saravanan, S. Sankar, S.    Gopalan, B. Nandagopal, Identification of immunogenic B-cell epitope    peptides of rubella virus E1 glycoprotein towards development of    highly specific immunoassays and/or vaccine. Conference Abstract,    (2019).-   7. U. S. F. Tambunan, F. R. P. Sipahutar, A. A. Parikesit, D.    Kerami, Vaccine Design for H5N1 Based on B- and T-cell Epitope    Predictions. Bioinform Biol Insights 10, 27-35 (2016).

Example 9—In Vivo Delivery of PE Agents

Precise genome targeting technologies using the CRISPR/Cas9 system haverecently been explored in a wide range of applications, including genetherapy. A major limitation to the application of Cas9 and Cas9-basedgenome-editing agents in gene therapy is the size of Cas9 (>4 kb),impeding its efficient delivery via recombinant adeno-associated virus(rAAV). Recently-developed fusions of Cas9 to reverse transcriptases(“Prime editors”) represent a novel genome editing technology thatpossesses a number of advantages over existing genome editing methods,including the ability to install any single nucleotide substitution, andto insert or delete any arbitrarily-defined short (<˜20) stretch ofnucleotides in a site-specific manner. As such, this method enablesediting of human pathogenic variants that have been intractable tocorrection previously. The delivery of prime editing reagents couldenable correction of genetic sequences that cause human disease, orallow for the installation of disease-preventing gene variants.

This invention describes methods for delivering prime editors into cellsin vitro and in vivo. Prime editors have been developed andcharacterized solely in cultured cells. No known method can deliverprime editors in vivo. The presently disclosed methods for deliveringprime editors via rAAV or pre-assembled ribonucleoprotein (RNP)complexes will overcome several barriers to in vivo delivery. Forexample, the DNA encoding prime editors is larger than the rAAVpackaging limit, and so requires special solutions. One such solution isformulating the editor fused to split intein pairs that are packagedinto two separate rAAV particles that, when co-delivered to a cell,reconstitute the functional editor protein. Several other specialconsiderations to account for the unique features of prime editing aredescribed, including the optimization of second-site nicking targets andproperly packaging prime editors into virus vectors, includinglentiviruses and rAAV.

Distinguishing features include using ribonucleoprotein (RNP) deliveryformulations, prime editors and nearby nicking targets can bepre-complexed with their specific sgRNA/PEgRNA. This will enhance therange of possible targetable sites and allow for greater optimization ofediting efficiency relative to current data that has used DNA delivery.Using either RNP or mRNA delivery formulations, variant Cas proteins canbe used that each complex with their own guide RNA variant. This willalso allow for a greater diversity of potential nicking loci, so it isexpected that optimization can be achieved for greater efficiency in anygiven application. Using RNP, it is expected to increase editingspecificity base on previous RNP reports (Rees et al., 2017). This wouldreduce off-target prime editing. Potential architectures for splittingprime editors into two AAV vectors for delivery in vivo or ex vivo aredescribed. Packaging prime editor into a dual AAV system requiresoptimization of design considerations including split sites,reconstitution methods (such as inteins), and guide expressionarchitecture. Using a mixture of virus and RNP for delivery of primeeditor, it is expected that editing will be controlled over time sinceRNP eventually degrades in vivo which will stop prime editing after RNPis no longer supplied.

Prime editor ribonucleoprotein (RNP), mRNA with prime editor guide RNA,or DNA can be packaged into lipid nanoparticles, rAAV, or lentivirus andinjected, ingested, or inhaled to alter genomic DNA in vivo and ex vivo,including for the purposes of establishing animal models of humandisease, testing therapeutic and scientific hypotheses in animal modelsof human disease, and treating disease in humans.

Prime editors could feasibly be used to correct a large fraction of allgenetic diseases (˜89% of pathogenic human genetic variants in Clinvar),if suitable means of delivery into relevant cell types in vivo aredeveloped. Blood diseases, retinal diseases, and liver diseases are themost likely first applications due to established delivery systems forother reagents. AAV capsids, other evolved or engineered viral vectors,and lipid nanoparticle formulations would need to be used in combinationwith this invention.

In certain embodiments, one or more of the prime editor domains (e.g.,the napDNAbp domain or the RT domain) could be engineered with an inteinsequence.

Example 10—Use of PE to Identify Off-Target Editing

There are currently no described methods to detect off-target editingwith prime editors (prime editing itself has not been published yet).These methods would allow a researcher to identify potential sites ofoff-target editing using prime editors, which would be importantconsiderations were this technique used to treat genetic disease inpatients.

Methods described here could also be useful to identify off-targets ofCas nucleases. These off-targets have previously been identified usingBLESS, Guide-Seq, CIRCLE-Seq, and Digenome-Seq. However, this method isadvantageous in the sensitivity and simplicity of the process.

The key concept of this aspect is the idea of using prime editing toinsert an adapter sequence or primer binding site, templated from aPEgRNA, to enable the rapid identification of genomic off-targetmodification sites of Cas nucleases or prime editors.

No method to identify in an unbiased manner prime editing off-targetsites is known. This method is distinguished from other techniques thatidentify nuclease off-target sites because the adapter sequence isinserted in the same event as DNA binding and nicking, simplifying thedownstream processing.

The present invention includes identification of off-target editingsites when editing inside a living cell, in tissue culture or animalmodels (see FIG. 33 ). To conduct this method, a PEgRNA is generatedthat has an identical protospacer to the final desired editor (and, iflooking at prime editing off-targets, an identical primer-binding sitesequence to the final desired editor), but includes the necessarysequences to install an adapter or primer binding site after reversetranscription by prime editing. In vivo editing is conducted using aprime editor or RT-fused nuclease, and isolate genomic DNA. The genomicDNA is fragmented by enzymatic or mechanical means and append adifferent adapter to sites of DNA fragmentation. PCR is used to amplifyfrom one adapter to the adapter installed via PEgRNA. The resultingproduct is deep-sequenced to identify all modified sites.

The invention also includes identification of off-target editing sitesusing in vitro modification of genomic DNA (see FIG. 33 ). To conductthis method, RNP of purified prime editor protein and a PEgRNA isassembled that will install an adapter or primer binding sequence, butis otherwise the same as the PEgRNA of interest. This RNP is incubatedwith extracted genomic DNA before or after fragmentation of the DNA andattachment of different adapters to sites of DNA breaks. PCR is used toamplify from fragmented site to the adapter that was installed with PE.Deep sequence to identify sites of modification. This in vitro editingmethod should enhance the sensitivity of detection, because cellular DNArepair will never eliminate the reverse-transcribed DNA adapter added bythe prime editor.

These methods could be used to identify off-target editing for any primeeditor, or any genome editor that uses a guideRNA to recognize a targetcut site (most Cas nucleases).

These methods could be applied to all genetic diseases for which genomeeditors are considered for use in treatment.

Example 11—Use of PE to Enable Chemical-Induced Dimerization of TargetProteins In Vivo

The prime editors described herein may also be used to placedimerization-induced biological processes, such as receptor signaling,under control of a convenient small-molecule drug by the genomicintegration of genes encoding small-molecule binding proteins with primeediting is described herein. Using the prime editors described herein,the gene sequence encoding a small-molecule binding protein may beinserted within a gene encoding a target protein of interest in a livingcell or patient. This edit alone should have no physiological effect.Upon administration of the small-molecule drug, which typically is adimeric small molecule that can simultaneously bind to two drug-bindingprotein domains each of which is fused to a copy of the target protein,the small-molecule induces dimerization of the targeted protein. Thistarget protein dimerization event then induces a biological signalingevent, such as erythropoiesis or insulin signaling.

Example 12—Prime Editing: Highly Versatile and PreciseSearch-and-Replace Genome Editing in Human Cells without Double-StrandedDNA Breaks

Current genome editing methods can disrupt, delete, or insert targetgenes with accompanying byproducts of double-stranded DNA breaks usingprogrammable nucleases, and install the four transition point mutationsat target loci using base editors. Small insertions, small deletions,and the eight transversion point mutations, however, collectivelyrepresent most pathogenic genetic variants but cannot be correctedefficiently and without an excess of byproducts in most cell types.Described herein is prime editing, a highly versatile and precise genomeediting method that directly writes new genetic information into aspecified DNA site using a catalytically impaired Cas9 fused to anengineered reverse transcriptase, programmed with an engineered primeediting guide RNA (PEgRNA) that both specifies the target site andencodes the desired edit. Greater than 175 distinct edits in human cellswere performed to establish that prime editing can make targetedinsertions, deletions, all 12 possible types of point mutations, andcombinations thereof efficiently (typically 20-60%, up to 77% inunsorted cells) and with low byproducts (typically 1-10%), withoutrequiring double-stranded breaks or donor DNA templates. Prime editingwas applied in human cells to correct the primary genetic causes ofsickle cell disease (requiring an A·T-to-T·A transversion in HBB) andTay-Sachs disease (requiring a 4-base deletion in HEXA), in both casesefficiently reverting the pathogenic genomic alleles to wild-type withminimal byproducts. Prime editing was also used to create human celllines with these pathogenic HBB transversion and HEXA insertionmutations, to install the G127V mutation in PRNP that confers resistanceto prion disease (requiring a G·C-to-T·A transversion), and toefficiently insert a His6 tag, a FLAG epitope tag, and an extended LoxPsite into target loci in human cells. Prime editing offers efficiencyand product purity advantages over HDR, and complementary strengths andweaknesses compared to base editing. Consistent with itssearch-and-replace mechanism, which requires three distinct base-pairingevents, prime editing is much less prone to off-target DNA modificationat known Cas9 off-target sites than Cas9. Prime editing substantiallyexpands the scope and capabilities of genome editing, and in principlecan correct ˜89% of known pathogenic human genetic variants.

The ability to make virtually any targeted change in the genome of anyliving cell or organism is a longstanding aspiration of the lifesciences. Despite rapid advances in genome editing technologies, themajority of the >75,000 known human genetic variants associated withdiseases¹¹¹ cannot be corrected or installed in most therapeuticallyrelevant cells (FIG. 38A). Programmable nucleases such as CRISPR-Cas9make double-stranded DNA breaks (DSBs) that can disrupt genes byinducing mixtures of insertions and deletions (indels) at targetsites¹¹²⁻¹¹⁴. Nucleases can also be used to delete targetgenes^(115,116), or insert exogenous genes¹¹⁷-¹¹⁹, throughhomology-independent processes. Double-stranded DNA breaks, however, arealso associated with undesired outcomes including complex mixtures ofproducts, translocations¹²⁰, and p53 activation^(121,122). Moreover, thevast majority of pathogenic alleles differ from their non-pathogeniccounterparts by small insertions, deletions, or base substitutions thatrequire much more precise editing technologies to correct (FIG. 38A).Homology-directed repair (HDR) stimulated by nuclease-induced DSBs¹²³has been widely used to install a variety of precise DNA changes. HDR,however, relies on exogenous donor DNA repair templates, typicallygenerates an excess of indel byproducts from end-joining repair of DSBs,and is inefficient in most therapeutically relevant cell types (T cellsand some stem cells being important exceptions)^(124,125). Whileenhancing the efficiency and precision of DSB-mediated genome editingremains the focus of promising efforts¹²⁶⁻¹³⁰, these challengesnecessitate the exploration of alternative precision genome editingstrategies.

Base editing can efficiently install or correct the four types oftransition mutations (C to T, G to A, A to G, and T to C) withoutrequiring DSBs in a wide variety of cell types and organisms, includingmammals¹²⁸⁻¹³¹, but cannot currently achieve any of the eighttransversion mutations (C to A, C to G, G to C, G to T, A to C, A to T,T to A, and T to G), such as the T·A-to-A·T mutation needed to directlycorrect the most common cause of sickle cell disease (HBB E6V)¹³². Inaddition, no DSB-free method has been reported to perform targetdeletions, such as the removal of the 4-base duplication that causesTay-Sachs disease (HEXA 1278+TATC)¹³³, or targeted insertions, such asthe precise 3-base insertion required to directly correct the mostcommon cause of cystic fibrosis (CFTR ΔF508)¹³⁴. Targeted transversionpoint mutations, insertions, and deletions thus are difficult to installor correct efficiently and without excess byproducts in most cell types,even though they collectively account for most known pathogenic alleles(FIG. 38A).

Described herein is the development of prime editing, a new“search-and-replace” genome editing technology that mediates targetedinsertions, deletions, and all 12 possible base-to-base conversions attargeted loci in human cells without requiring double-stranded DNAbreaks, or donor DNA templates. Prime editors, initially exemplified byPE1, use a reverse transcriptase fused to a programmable nickase and aprime editing extended guide RNA (PEgRNA) to directly copy geneticinformation from the extension on the PEgRNA into the target genomiclocus. A second-generation prime editor (PE2) uses an engineered reversetranscriptase to substantially increase editing efficiencies withminimal (typically <2%) indel formation, while a third-generation PE3system adds a second guide RNA to nick the non-edited strand, therebyfavoring replacement of the non-edited strand and further increasingediting efficiency, typically, to about 20-50% in human cells with about1-10% indel formation. PE3 offers far fewer byproducts and higher orsimilar efficiency compared to optimized Cas9 nuclease-initiated HDR,and offers complementary strengths and weaknesses compared tocurrent-generation base editors.

PE3 was applied at genomic loci in human HEK293T cells to achieveefficient conversion of HBB E6V to wild-type HBB, deletion of theinserted TATC to restore HEXA 1278+TATC to wild-type HEXA, installationin PRNP of the G127V mutation that confers resistance to priondisease¹³⁵ (requiring a G·C-to-T·A transversion), and targeted insertionof a His₆ tag (18 bp), FLAG epitope tag (24 bp), and extended LoxP sitefor Cre-mediated recombination (44 bp). Prime editing was alsosuccessful in three other human cell lines, as well as in post-mitoticprimary mouse cortical neurons, with varying efficiencies. Due to a highdegree of flexibility in the distance between the initial nick andlocation of the edit, prime editing is not substantially constrained bythe PAM requirement of Cas9 and in principle can target the vastmajority of genomic loci. Off-target prime editing is much rarer thanoff-target Cas9 editing at known Cas9 off-target loci, likely due to therequirement of three distinct DNA base pairing events in order forproductive prime editing to take place. By enabling precise targetedinsertions, deletions, and all 12 possible classes of point mutations ata wide variety of genomic loci without the need for DSBs or donor DNAtemplates, prime editing has the potential to advance the study andcorrection of many gene variants.

Results

Strategy for Transferring Information from an Extended Guide RNA into aTarget DNA Locus

Cas9 targets DNA using a guide RNA containing a spacer sequence thathybridizes to the target DNA site^(112-114,136,137). The aim was toengineer guide RNAs to both specify the DNA target as in natural CRISPRsystems^(138,139), and also to contain new genetic information thatreplaces the corresponding DNA nucleotides at the target locus. Thedirect transfer of genetic information from an extended guide RNA into aspecified DNA site, followed by replacement of the original uneditedDNA, in principle could provide a general means of installing targetedDNA sequence changes in living cells, without dependence on DSBs ordonor DNA templates. To achieve this direct information transfer, theaim was to use genomic DNA, nicked at the target site to expose a3′-hydroxyl group, to prime the reverse transcription of the geneticinformation from an extension on the engineered guide RNA (hereafterreferred to as the prime editing guide RNA, or PEgRNA) directly into thetarget site (FIG. 38A).

These initial steps of nicking and reverse transcription, which resemblemechanisms used by some natural mobile genetic elements¹⁴⁰, result in abranched intermediate with two redundant single-stranded DNA flaps onone strand: a 5′ flap that contains the unedited DNA sequence, and a 3′flap that contains the edited sequence copied from the PEgRNA (FIG.38B). To achieve a successful edit, this branched intermediate must beresolved so that the edited 3′ flap replaces the unedited 5′ flap. Whilehybridization of the 5′ flap with the unedited strand is likely to bethermodynamically favored since the edited 3′ flap can make fewer basepairs with the unedited strand, 5′ flaps are the preferred substrate forstructure-specific endonucleases such as FEN1¹⁴¹, which excises 5′ flapsgenerated during lagging-strand DNA synthesis and long-patch baseexcision repair. It was reasoned that preferential 5′ flap excision and3′ flap ligation could drive the incorporation of the edited DNA strand,creating heteroduplex DNA containing one edited strand and one uneditedstrand (FIG. 38B).

Permanent installation of the edit could arise from subsequent DNArepair that resolves the mismatch between the two DNA strands in amanner that copies the information in the edited strand to thecomplementary DNA strand (FIG. 38C). Based on a similar strategydeveloped to maximize the efficiency of DNA base editing¹³¹⁻¹³³, it wasenvisioned that nicking the non-edited DNA strand, far enough from thesite of the initial nick to minimize double-strand break formation,might bias DNA repair to preferentially replace the non-edited strand.

Validation of Prime Editing Steps In Vitro and in Yeast Cells

Following cleavage of the PAM-containing DNA strand by the RuvC nucleasedomain of Cas9, the PAM-distal fragment of this strand can dissociatefrom otherwise stable Cas9:sgRNA:DNA complexes¹⁴³. It was hypothesizedthat the 3′ end of this liberated strand might be sufficientlyaccessible to prime DNA polymerization. Guide RNA engineeringefforts¹⁴⁴⁻¹⁴⁶ and crystal structures of Cas9:sgRNA:DNA complexes¹⁴⁷⁻¹⁴⁹suggest that the 5′ and 3′ termini of the sgRNA can be extended withoutabolishing Cas9:sgRNA activity. PEgRNAs were designed by extendingsgRNAs to include two critical components: a primer binding site (PBS)that allows the 3′ end of the nicked DNA strand to hybridize to thePEgRNA, and a reverse transcriptase (RT) template containing the desirededit that would be directly copied into the genomic DNA site as the 3′end of the nicked DNA strand is extended across the RNA template by apolymerase (FIG. 38C).

These hypotheses were tested in vitro using purified S. pyogenes Cas9protein. A series of PEgRNA candidates were constructed by extendingsgRNAs on either terminus with a PBS sequence (5 to 6 nucleotides, nt)and an RT template (7 to 22 nt). It was confirmed that 5′-extendedPEgRNAs direct Cas9 binding to target DNA, and that both 5′-extendedPEgRNAs and 3′-extended PEgRNAs support Cas9-mediated target nicking invitro and DNA cleavage activities in mammalian cells (FIGS. 44A-44C).These candidate PEgRNA designs were tested using pre-nicked5′-Cy5-labeled dsDNA substrates, catalytically dead Cas9 (dCas9), and acommercial variant of Moloney murine leukemia virus (M-MLV) reversetranscriptase (FIG. 44D). When all components were present, efficientconversion of the fluorescently labeled DNA strand into longer DNAproducts with gel mobilities, consistent with reverse transcriptionalong the RT template, (FIG. 38D, FIGS. 44D-44E) was observed. Productsof desired length were formed with either 5′-extended or 3′-extendedPEgRNAs (FIGS. 38D-38E). Omission of dCas9 led to nick translationproducts derived from reverse transcriptase-mediated DNA polymerizationon the DNA template, with no PEgRNA information transfer (FIG. 38D). NoDNA polymerization products were observed when the PEgRNA was replacedby a conventional sgRNA, confirming the necessity of the PBS and RTtemplate components of the PEgRNA (FIG. 38D). These results demonstratethat Cas9-mediated DNA melting exposes a single-stranded R-loop that, ifnicked, is competent to prime reverse transcription from either a5′-extended or 3′-extended PEgRNA.

Next, non-nicked dsDNA substrates were tested with a Cas9 nickase (H840Amutant) that exclusively nicks the PAM-containing strand¹¹². In thesereactions, 5′-extended PEgRNAs generated reverse transcription productsinefficiently, possibly due to impaired Cas9 nickase activity (FIG.44F). However, 3′-extended PEgRNAs enabled robust Cas9 nicking andefficient reverse transcription (FIG. 38E). The use of 3′-extendedPEgRNAs generated only a single apparent product, despite the potential,in principle, for reverse transcription to terminate anywhere within theremainder of the PEgRNA. DNA sequencing of the products of reactionswith Cas9 nickase, RT, and 3′-extended PEgRNAs revealed that thecomplete RT template sequence was reverse transcribed into the DNAsubstrate (FIG. 44G). These experiments established that 3′-extendedPEgRNAs can template the reverse transcription of new DNA strands whileretaining the ability to direct Cas9 nickase activity.

To evaluate the eukaryotic cell DNA repair outcomes of 3′ flaps producedby PEgRNA-programmed reverse transcription in vitro, DNA nicking andreverse transcription using PEgRNAs, Cas9 nickase, and RT in vitro onreporter plasmid substrates were performed, and the reaction productswere then transformed into yeast (S. cerevisiae) cells (FIG. 45A).Encouragingly, when plasmids were edited in vitro with 3′-extendedPEgRNAs encoding a T·A-to-A·T transversion that corrects the prematurestop codon, 37% of yeast transformants expressed both GFP and mCherryproteins (FIG. 38F, FIG. 45C). Consistent with the results in FIG. 38Eand FIG. 44F, editing reactions carried out in vitro with 5′-extendedPEgRNAs yielded fewer GFP and mCherry double-positive colonies (9%) thanthose with 3′-extended PEgRNAs (FIG. 38F and FIG. 45D). Productiveediting was also observed using 3′-extended PEgRNAs that insert a singlenucleotide (15% double-positive transformants) or delete a singlenucleotide (29% double-positive transformants) to correct frameshiftmutations (FIG. 38F and FIGS. 45E-45F). DNA sequencing of editedplasmids recovered from double-positive yeast colonies confirmed thatthe encoded transversion edit occurred at the desired sequence position(FIG. 45G). These results demonstrate that DNA repair in eukaryoticcells can resolve 3′ DNA flaps arising from prime editing to incorporateprecise DNA edits including transversions, insertions, and deletions.

Design of Prime Editor 1 (PE1)

Encouraged by the results in vitro and in yeast, a prime editing systemwith a minimum number of components capable of editing genomic DNA inmammalian cells was sought for development. It was hypothesized that3′-extended PEgRNAs (hereafter referred to simply as PEgRNAs, FIG. 39A)and direct fusions of Cas9 H840A to reverse transcriptase via a flexiblelinker may constitute a functional two-component prime editing system.HEK293T (immortalized human embryonic kidney) cells were transfectedwith one plasmid encoding a fusion of wild-type M-MLV reversetranscriptase to either terminus of Cas9 H840A nickase as well as asecond plasmid encoding a PEgRNA. Initial attempts led to no detectableT·A-to-A·T conversion at the HEK3 target locus.

Extension of the PBS in the PEgRNA to 8-15 bases (FIG. 39A), however,led to detectable T·A-to-A·T editing at the HEK3 target site (FIG. 39B),with higher efficiencies for prime editor constructs in which the RT wasfused to the C-terminus of Cas9 nickase (3.7% maximal T·A-to-A·Tconversion with PBS lengths ranging from 8-15 nt) compared to N-terminalRT-Cas9 nickase fusions (1.3% maximal T·A-to-A·T conversion) (FIG. 39B;all mammalian cell data herein reports values for the entire treatedcell population, without selection or sorting, unless otherwisespecified). These results suggest that wild-type M-MLV RT fused to Cas9requires longer PBS sequences for genome editing in human cells comparedto what is required in vitro using the commercial variant of M-MLV RTsupplied in trans. This first-generation wild-type M-MLV reversetranscriptase fused to the C-terminus of Cas9 H840A nickase wasdesignated as PE1.

The ability of PE1 to precisely introduce transversion point mutationsat four additional genomic target sites specified by the PEgRNA (FIG.39C) was tested. Similar to editing at the HEK3 locus, efficiency atthese genomic sites was dependent on PBS length, with maximal editingefficiencies ranging from 0.7-5.5% (FIG. 39C). Indels from PE1 were low,averaging 0.2i0.1% for the five sites under conditions that maximizedeach site's editing efficiency (FIG. 46A). PE1 was also able to installtargeted insertions and deletions, exemplified by a single-nucleotidedeletion (4.0% efficiency), a single-nucleotide insertion (9.7%), and athree-nucleotide insertion (17%) at the HEK3 locus (FIG. 39C). Theseresults establish the ability of PE1 to directly install targetedtransversions, insertions, and deletions without requiringdouble-stranded DNA breaks or DNA templates.

Design of Prime Editor 2 (PE2)

While PE1 can install a variety of edits at several loci in HEK293Tcells, editing efficiencies were generally low (typically ≤5%) (FIG.39C). It was hypothesized that engineering the reverse transcriptase inPE1 might improve the efficiency of DNA synthesis within the uniqueconformational constraints of the prime editing complex, resulting inhigher genome editing yields. M-MLV RT mutations have been previouslyreported that increase enzyme thermostability^(150,151),processivity¹⁵⁰, and DNA:RNA heteroduplex substrate affinity¹⁵², andthat inactivate RNaseH activity¹⁵³. 19 PE1 variants were constructedcontaining a variety of reverse transcriptase mutations to evaluatetheir prime editing efficiency in human cells.

First, a series of M-MLV RT variants that previously emerged fromlaboratory evolution for their ability to support reverse transcriptionat elevated temperatures¹⁵⁰ were investigated. Successive introductionof three of these amino acid substitutions (D200N, L603W, and T330P)into M-MLV RT, hereafter referred to as M3, led to a 6.8-fold averageincrease in transversion and insertion editing efficiency across fivegenomic loci in HEK293T cells compared to that of PE1 (FIGS. 47A-47S).

Next, in combination with M3, additional reverse transcriptase mutationsthat were previously shown to enhance binding to template:PBS complex,enzyme processivity, and thermostability¹⁵² were tested. Among the 14additional mutants analyzed, a variant with T306K and W313Fsubstitutions, in addition to the M3 mutations, improved editingefficiency an additional 1.3-fold to 3.0-fold compared to M3 for sixtransversion or insertion edits across five genomic sites in human cells(FIGS. 47A-47S). This pentamutant of M-MLV reverse transcriptaseincorporated into the PE1 architecture (Cas9 H840A-M-MLV RT (D200N L603WT330P T306K W313F)) is hereafter referred to as PE2.

PE2 installs single-nucleotide transversion, insertion, and deletionmutations with substantially higher efficiency than PE1 (FIG. 39C), andis compatible with shorter PBS PEgRNA sequences (FIG. 39C), consistentwith an enhanced ability to productively engage transient genomicDNA:PBS complexes. On average, PE2 led to a 1.6- to 5.1-fold improvementin prime editing point mutation efficiency over PE1 (FIG. 39C), and insome cases dramatically improved editing yields up to 46-fold (FIG. 47Fand FIG. 47I). PE2 also effected targeted insertions and deletions moreefficiently than PE1, achieving the targeted insertion of the 24-bp FLAGepitope tag at the HEK3 locus with 4.5% efficiency, a 15-foldimprovement over the efficiency of installing this insertion with PE1(FIG. 47D), and mediated a 1-bp deletion in HEK3 with 8.6% efficiency,2.1-fold higher than that of PE1 (FIG. 39C). These results establish PE2as a more efficient prime editor than PE1.

Optimization of PEgRNA Features

The relationship between PEgRNA architecture and prime editingefficiency was systematically probed at five genomic loci in HEK293Tcells with PE2 (FIG. 39C). In general, priming sites with lower GCcontent required longer PBS sequences (EMX1 and RNF2, containing 40% and30% GC content, respectively, in the first 10 nt upstream of the nick),whereas those with greater GC content supported prime editing withshorter PBS sequences (HEK4 and FANCF, containing 80% and 60% GCcontent, respectively, in the first 10 nt upstream of the nick) (FIG.39C), consistent with the energetic requirements for hybridization ofthe nicked DNA strand to the PEgRNA PBS. No PBS length or GC contentlevel was strictly predictive of prime editing efficiency, and otherfactors such as secondary structure in the DNA primer or PEgRNAextension may also influence editing activity. It is recommended tostart with a PBS length of ˜13 nt for a typical target sequence, andexploring different PBS lengths if the sequence deviates from ˜40-60% GCcontent. When necessary, optimal PBS sequences should be determinedempirically.

Next, the performance determinants of the RT template portion of thePEgRNA were studied. PEgRNAs with RT templates ranging from 10-20 nt inlength were systemically evaluated at five genomic target sites usingPE2 (FIG. 39D) and with longer RT templates as long as 31 nt at threegenomic sites (FIGS. 48A-48C). As with PBS length, RT template lengthalso could be varied to maximize prime editing efficiency, although ingeneral many RT template lengths >10 nt long support more efficientprime editing (FIG. 39D). Since some target sites preferred longer RTtemplates (>15 nt) to achieve higher editing efficiencies (FANCF, EMX1),while other loci preferred short RT templates (HEK3, HEK4) (FIG. 39D),it is recommend both short and long RT templates be tested whenoptimizing a PEgRNA, starting with ˜10-16 nt.

Importantly, RT templates that place a C as the nucleotide adjacent tothe terminal hairpin of the sgRNA scaffold generally resulted in lowerediting efficiency compared to other PEgRNAs with RT templates ofsimilar length (FIGS. 48A-48C). Based on the structure of sgRNAs boundto Cas9^(148,149), it was speculated that the presence of a C as thefirst nucleotide of the 3′ extension of a canonical sgRNA can disruptthe sgRNA scaffold fold by pairing with G81, a nucleotide that nativelyforms a pi stack with Tyr 1356 in Cas9 and a non-canonical base pairwith sgRNA A68. Since many RT template lengths support prime editing, itis recommended to choose PEgRNAs in which the first base of the 3′extension (the last reverse-transcribed base of the RT template) is notC.

Design of Prime Editor 3 Systems (PE3 and PE3b)

While PE2 can transfer genetic information from the PEgRNA to the targetlocus more efficiently than PE1, the manner in which the cell resolvesthe resulting heteroduplex DNA created by one edited strand and oneunedited strand determines if the edit is durable. A previousdevelopment of base editing faced a similar challenge since the initialproduct of cytosine or adenine deamination is heteroduplex DNAcontaining one edited and one non-edited strand. To increase theefficiency of base editing, a Cas9 D10A nickase was used to introduce anick into the non-edited strand and to direct DNA repair to that strand,using the edited strand as a template^(129,130,142). To exploit thisprinciple to enhance prime editing efficiencies, a similar strategy ofnicking the non-edited strand using the Cas9 H840A nickase alreadypresent in PE2 and a simple sgRNA to induce preferential replacement ofthe non-edited strand by the cell (FIG. 40A) was tested. Since theedited DNA strand was also nicked to initiate prime editing, a varietyof sgRNA-programmed nick locations were tested on the non-edited strandto minimize the production of double-stranded DNA breaks that lead toindels.

This PE3 strategy was first tested at five genomic sites in HEK293Tcells by screening sgRNAs that induce nicks located 14 to 116 bases fromthe site of the PEgRNA-induced nick, either 5′ or 3′ of the PAM. In fourof the five sites tested, nicking the non-edited strand increased theamount of indel-free prime editing products compared to the PE2 systemby 1.5- to 4.2-fold, to as high as 55% (FIG. 40B). While the optimalnicking position varied depending on the genomic site, nicks positioned3′ of the PAM (positive distances in FIG. 40B) approximately 40-90 bpfrom the PEgRNA-induced nick generally produced favorable increases inprime editing efficiency (averaging 41%) without excess indel formation(6.8% average indels for the sgRNA resulting in the highest editingefficiency for each of the five sites tested) (FIG. 40B). As expected,at some sites, placement of the non-edited strand nick within 40 bp ofthe PEgRNA-induced nick led to large increases in indel formation up to22% (FIG. 40B), presumably due to the formation of a double-strand breakfrom nicking both strands close together. At other sites, however,nicking as close as 14 bp away from the PEgRNA-induced nick producedonly 5% indels (FIG. 40B), suggesting that locus-dependent factorscontrol conversion of proximal dual nicks into double-strand DNA breaks.At one tested site (HEK4), complementary strand nicks either provided nobenefit or led to indel levels that surpassed editing efficiency (up to26%), even when placed at distances >70 bp from the PEgRNA-induced nick,consistent with an unusual propensity of the edited strand at that siteto be nicked by the cell, or to be ligated inefficiently. It isrecommend to start with non-edited strand nicks approximately 50 bp fromthe PEgRNA-mediated nick, and to test alternative nick locations ifindel frequencies exceed acceptable levels.

This model for how complementary strand nicking improved prime editingefficiency (FIG. 40A) predicted that nicking the non-edited strand onlyafter edited strand flap resolution could minimize the presence ofconcurrent nicks, decreasing the frequency of double-strand breaks thatgo on to form indels. To achieve temporal control over non-edited strandnicking, sgRNAs with spacer sequences that match the edited strand, butnot the original allele, were designed. Using this strategy, referred tohereafter as PE3b, mismatches between the spacer and the unedited alleleshould disfavor nicking by the sgRNA until after the editing event onthe PAM strand takes place. This PE3b approach was tested with fivedifferent edits at three genomic sites in HEK293T cells and comparedoutcomes to those achieved with PE2 and PE3 systems. In all cases, PE3bwas associated with substantially lower levels of indels compared to PE3(3.5- to 30-fold, averaging 12-fold lower indels, or 0.85%), without anyevident decrease in overall editing efficiency compared to PE3 (FIG.40C). Therefore, when the edit lay within a second protospacer, the PE3bsystem could decrease indels while still improving editing efficiencycompared to PE2, often to levels similar to those of PE3 (FIG. 40C).

Together, these findings established that PE3 systems (Cas9nickase-optimized reverse transcriptase+PEgRNA+sgRNA) improved editingefficiencies ˜3-fold compared with PE2 (FIGS. 40B-40C). PE3 wasaccompanied by wider ranges of indels than PE2, as expected given theadditional nicking activity of PE3. The use of PE3 is recommended whenprioritizing prime editing efficiency. When minimization of indels iscritical, PE2 offers ˜10-fold lower indel frequencies. When it ispossible to use a sgRNA that recognizes the installed edit to nick thenon-edited strand, the PE3b system can achieve PE3-like editing levelswhile greatly reducing indel formation.

To demonstrate the targeting scope and versatility of prime editing withPE3, the installation of all possible single nucleotide substitutionsacross the +1 to +8 positions (counting the first base 3′ of thePEgRNA-induced nick as position +1) of the HEK3 target site using PE3and PEgRNAs with 10-nucleotide RT templates (FIG. 41A) was explored.Collectively, these 24 distinct edits cover all four transitionmutations and all eight transversion mutations, and proceed with editingefficiencies (containing no indels) averaging 33±7.9% (ranging between14% and 48%), with an average of 7.5±1.8% indels.

Importantly, long-distance RT templates could also give rise toefficient prime editing with PE3. For example, using PE3 with a 34-nt RTtemplate, point mutations were installed at positions +12, +14, +17,+20, +23, +24, +26, +30, and +33 (12 to 33 bases from the PEgRNA-inducednick) in the HEK3 locus with an average of 36±8.7% efficiency and8.6±2.0% indels (FIG. 41B). Although edits beyond the +10 position atother loci were not attempted, other RT templates ≥30 nt at threealternative sites also support efficient editing (FIGS. 48A-C). Theviability of long RT templates enabled efficient prime editing fordozens of nucleotides from the initial nick site. Since an NGG PAM oneither DNA strand occurs on average every ˜8 bp, far less than maximumdistances between the edit and the PAM that support efficient primeediting, prime editing is not substantially constrained by theavailability of a nearby PAM sequence, in contrast with other precisiongenome editing methods^(125,142,154). Given the presumed relationshipbetween RNA secondary structure and prime editing efficiency, whendesigning PEgRNAs for long-range edits it is prudent to test RTtemplates of various lengths and, if necessary, sequence compositions(e.g., synonymous codons) to optimize editing efficiency.

To further test the scope and limitations of the PE3 system forintroducing transition and transversion point mutations, 72 additionaledits covering all 12 possible types of point mutations across sixadditional genomic target sites (FIG. 41C-41H) were tested. Overall,indel-free editing efficiency averaged 25±14%, while indel formationaveraged 8.3±7.5%. Since the PEgRNA RT template included the PAMsequence, prime editing could induce changes to the PAM sequence. Inthese cases, higher editing efficiency (averaging 39±9.7%) and lowerindel generation (averaging 5.0±2.9%) were observed (FIGS. 41A-41K,point mutations at positions +5 or +6). This increase in efficiency anddecrease in indel formation for PAM edits may arise from the inabilityof the Cas9 nickase to re-bind and nick the edited strand prior to therepair of the complementary strand. Since prime editing supportscombination edits with no apparent loss of editing efficiency, editingthe PAM, in addition to other desired changes, when possible, isrecommended.

Next, 14 targeted small insertions and 14 targeted small deletions atseven genomic sites using PE3 (FIG. 41I) were performed. Targeted 1-bpinsertions proceeded with an average efficiency of 32±9.8%, while 3-bpinsertions were installed with an average efficiency of 39±16%. Targeted1-bp and 3-bp deletions were also efficient, proceeding with an averageyield of 29±14% and 32±11%, respectively. Indel generation (beyond thetargeted insertion or deletion) averaged 6.8±5.4%. Since insertions anddeletions introduced between positions +1 and +6 alter the position orthe structure of the PAM, it was speculated that insertion and deletionedits in this range are typically more efficient due to the inability ofCas9 nickase to re-bind and nick the edited DNA strand prior to repairof the complementary strand, similar to point mutations that edit thePAM.

PE3 was also tested for its ability to mediate larger precise deletionsof 5 bp to 80 bp at the HEK3 site (FIG. 41J). Very high editingefficiencies (52 to 78%) were observed for 5-, 10-, and 15-bp deletionswhen using a 13-nt PBS and an RT template that contained 29, 24, or 19bp of homology to the target locus, respectively. Using a 26-nt RTtemplate supported a larger deletion of 25 bp with 72±4.2% efficiency,while a 20-nt RT template enabled an 80-bp deletion with an efficiencyof 52±3.8%. These targeted deletions were accompanied by indelfrequencies averaging 11±4.8% (FIG. 41J).

Finally, the ability of PE3 to mediate 12 combinations of multiple editsat the same target locus consisting of insertions and deletions,insertions and point mutations, deletions and point mutations, or twopoint mutations across three genomic sites was tested. These combinationedits were very efficient, averaging 55% of the target edit with 6.4%indels (FIG. 41K), and demonstrating the ability of prime editing tomake combinations of precision insertions, deletions, and pointmutations at individual target sites with high efficiency and low indelfrequencies.

Together, the examples in FIGS. 41A-41K represent 156 distincttransition, transversion, insertion, deletion, and combination editsacross seven human genomic loci. These findings establish theversatility, precision, and targeting flexibility of prime editing.

Prime Editing Compared with Base Editing

Current-generation cytidine base editors (CBEs) and adenine base editors(ABEs) can install C·G-to-T·A transition mutations and A·T-to-G·Ctransition mutations with high efficiency and low indels^(129,130,142).The application of base editing can be limited by the presence ofmultiple cytidine or adenine bases within the base editing activitywindow (typically ˜5-bp wide), which gives rise to unwanted bystanderedits^(129,130,142,155) or by the absence of a PAM positionedapproximately 15±2 nt from the target nucleotide^(142,156). It wasanticipated that prime editing could be particularly useful for preciseinstallation of transitions mutations without bystander edits, or whenthe lack of suitably positioned PAMs precludes favorable positioning thetarget nucleotide within the CBE or ABE activity window.

Prime editing and cytosine base editing was compared by editing threegenomic loci that contain multiple target cytidines in the canonicalbase editing window (protospacer positions 4-8, counting the PAM aspositions 21-23) using optimized CBEs¹⁵⁷ without nickase activity(BE2max) or with nickase activity (BE4max), or using the analogous PE2and PE3 prime editing systems. Among the nine total target cytosineswithin the base editing windows of the three sites, BE4max yielded2.2-fold higher average total C·G-to-T·A conversion than PE3 for basesin the center of the base editing window (protospacer positions 5-7,FIG. 42A). Likewise, non-nicking BE2max outperformed PE2 by 1.4-fold onaverage at these well-positioned bases (FIG. 42A). However, PE3outperformed BE4max by 2.7-fold, and PE2 outperformed BE2max by2.0-fold, for cytosines beyond the center of the base editing window(average editing of 40±17% for PE3 vs. 15±18% for BE4max, and 22±11% forPE2 vs. 11±13% for BE2max). Overall, indel frequencies for PE2 were verylow (averaging 0.86±0.47%), and for PE3 were similar to or modestlyhigher than that of BE4max (BE4max range: 2.5% to 14%; PE3 range: 2.5%to 21%) (FIG. 42B).

When comparing the efficiency of base editing to prime editing forinstallation of precise C·G-to-T·A edits (without any bystanderediting), the efficiency of prime editing greatly exceeded that of baseediting at the above sites, which like most genomic DNA sites, containmultiple cytosines within the ˜5-bp base editing window (FIG. 42C). Atthese sites, such as EMX1, which contains cytosines at protospacerpositions C5, C6, and C7, BE4max generated few products containing onlythe single target base pair conversion with no bystander edits. Incontrast, prime editing at this site could be used to selectivelyinstall a C·G-to-T·A edit at any position or combination of positions(C5, C6, C7, C5+C6, C6+C7, C5+C7, or C5+C6+C7) (FIG. 42C). All preciseone-base or two-base edits (that is, edits that do not modify any othernearby bases) were much more efficient with PE3 or PE2 than with BE4maxor BE2, respectively, while the three-base C·G-to-T·A edit was moreefficient with BE4max (FIG. 42C), reflecting the propensity of baseeditors to edit all target bases within the activity window. Takentogether, these results demonstrate that cytosine base editors canresult in higher levels of editing at optimally positioned target basesthan PE2 or PE3, but prime editing can outperform base editing atnon-optimally positioned target bases, and can edit with much higherprecision with multiple editable bases.

A·T-to-G·C editing was compared at two genomic loci by an optimizednon-nicking ABE (ABEmax¹⁵² with a dCas9 instead of a Cas9 nickase,hereafter referred to as ABEdmax) versus PE2, and by the optimizednicking adenine base editor ABEmax versus PE3. At a site that containstwo target adenines in the base editing window (HEK3), ABEs were moreefficient than PE2 or PE3 for conversion of A5, but PE3 was moreefficient for conversion of A8, which lies at the edge of the ABEmaxediting window (FIG. 42D). When comparing the efficiency of precisionedits in which only a single adenine is converted, PE3 outperformedABEmax at both A5 and A8 (FIG. 42E). Overall, ABEs produced far fewerindels at HEK3 than prime editors (0.19±0.02% for ABEdmax vs. 1.5±10.46%for PE2, and 0.53±0.16% for ABEmax vs. 11±2.3% for PE3, FIG. 42F). AtFANCF, in which only a single A is present within the base editingwindow, ABE2 and ABEmax outperformed their prime editing counterparts intotal target base pair conversion by 1.8- to 2.9-fold, with virtuallyall edited products from both base editing and prime editing containingonly the precise edit (FIGS. 42D-42E). As with the HEK3 site, ABEsproduced far fewer indels at the FANCF site (FIG. 42F).

Collectively, these results indicate that base editing and prime editingoffer complementary strengths and weaknesses for making targetedtransition mutations. For cases in which a single target nucleotide ispresent within the base editing window, or when bystander edits areacceptable, current base editors are typically more efficient and resultin fewer indels than prime editors. When multiple cytosines or adeninesare present and bystander edits are undesirable, or when target basesare poorly positioned for base editing relative to available PAMs, primeeditors offer substantial advantages.

Off-Target Prime Editing

To result in productive editing, prime editing requires targetlocus:PEgRNA spacer complementary for the Cas9 domain to bind, targetlocus:PEgRNA PBS complementarity for PEgRNA-primed reverse transcriptionto initiate, and target locus:reverse transcriptase productcomplementarity for flap resolution. It was hypothesized that thesethree distinct DNA hybridization requirements may minimize off-targetprime editing compared to that of other genome editing methods. To testthis possibility, HEK293T cells were treated with PE3 or PE2 and 16total PEgRNAs designed to target four on-target genomic loci, with Cas9and the four corresponding sgRNAs targeting the same protospacers, orwith Cas9 and the same 16 PEgRNAs. These four target loci were chosenbecause each has at least four well-characterized off-target sites forwhich Cas9 and the corresponding on-target sgRNA in HEK293T cells isknown to cause substantial off-target DNA modification^(118,159).Following treatment, the four on-target loci and the top four known Cas9off-target sites for each on-target spacer, were sequenced, for a totalof 16 off-target sites (Table 1).

Consistent with previous studies¹¹⁸, Cas9 and the four target sgRNAsmodified all 16 of the previously reported off-target loci (FIG. 42G).Cas9 off-target modification efficiency among the four off-target sitesfor the HEK3 target locus averaged 16%. Cas9 and the sgRNA targetingHEK4 resulted in an average of 60% modification of the four tested knownoff-target sites. Likewise, off-target sites for EMX1 and FANCF weremodified by Cas9:sgRNA at an average frequency of 48% and 4.3%,respectively (FIG. 42G). It was noted that PEgRNAs with Cas9 nucleasemodified on-target sites at similar (1- to 1.5-fold lower) efficiency onaverage compared to sgRNAs, while PEgRNAs with Cas9 nuclease modifiedoff-target sites at ˜4-fold lower average efficiency than sgRNAs.

Strikingly, PE3 or PE2 with the same 16 tested PEgRNAs containing thesefour target spacers resulted in much lower off-target editing (FIG.42H). Of the 16 sites known to undergo off-target editing by Cas9+sgRNA,PE3+PEgRNAs or PE2+PEgRNAs resulted in detectable off-target primeediting at only 3 of 16 off-target sites, with only 1 of 16 showingoff-target editing efficiency ≥1% (FIG. 42H). Average off-target primeediting for the PEgRNAs targeting HEK3, HEK4, EMX1, and FANCF at these16 known Cas9 off-target sites was <0.1%, <2.2±5.2%, <0.1%, and<0.13±0.11%, respectively (FIG. 42H). Notably, at the HEK4 off-target 3site that Cas9+PEgRNA1 edits with 97% efficiency, PE2+PEgRNA1 results inonly 0.7% off-target editing despite sharing the same spacer sequence,demonstrating how the two additional DNA hybridization events requiredfor prime editing compared to Cas9 editing can greatly reduce off-targetediting. Taken together, these results suggest that PE3 and PEgRNAsinduce much lower off-target DNA editing in human cells than Cas9 andsgRNAs that target the same protospacers.

Reverse transcription of 3′-extended PEgRNAs in principle can proceedinto the guide RNA scaffold. If the resulting 3′ flap, despite a lack ofcomplementary at its 3′ end with the unedited DNA strand, isincorporated into the target locus, the outcome is insertion of PEgRNAscaffold nucleotides that contributes to indel frequency. We analyzedsequencing data from 66 PE3-mediated editing experiments at four loci inHEK293T cells and observed PEgRNA scaffold insertion at a low frequency,averaging 1.7±1.5% total insertion of any number of PEgRNA scaffoldnucleotides (FIGS. 56A-56D). It is speculated that inaccessibility ofthe guide RNA scaffold to the reverse transcriptase due to Cas9 domainbinding, as well as cellular excision during flap resolution of themismatched 3′ end of the 3′ flap that results from PEgRNA scaffoldreverse transcription, minimizes products that incorporate PEgRNAscaffold nucleotides. While such events are rare, future efforts toengineer PEgRNAs or prime editor proteins that minimize PEgRNA scaffoldincorporation may further decrease indel frequencies.

Deaminases in some base editors can act in a Cas9-independent manner,resulting in low-level but widespread off-target DNA editing amongfirst-generation CBEs (but not ABEs)¹⁶⁰⁻¹⁶² and off-target RNA editingamong first-generation CBEs and ABEs¹⁶³⁻¹⁶⁵, although newer CBE and ABEvariants with engineered deaminases greatly reduce Cas9-independentoff-target DNA and RNA editing¹⁶³⁻¹⁶⁵. Prime editors lackbase-modification enzymes such as deaminases, and therefore have noinherent ability to modify DNA or RNA bases in a Cas9-independentmanner.

While the reverse transcriptase domain in prime editors in principlecould process properly primed RNA or DNA templates in cells, it wasnoted that retrotransposons such as those in the LINE-1 family¹⁶⁶,endogenous retroviruses^(167,168), and human telomerase all providedactive endogenous human reverse transcriptases. Their natural presencein human cells suggests that reverse transcriptase activity itself isnot substantially toxic. Indeed, no PE3-dependent differences wereobserved in HEK293T cell viability compared to that of controlsexpressing dCas9, Cas9 H840A nickase, or PE2 with R110S+K103L (PE2-dRT)mutations that inactivate the reverse transcriptase¹⁶⁹ (FIGS. 49A-49B).

The above data and analyses notwithstanding, additional studies areneeded to assess off-target prime editing in an unbiased, genome-widemanner, as well as to characterize the extent to which the reversetranscriptase variants in prime editors, or prime editing intermediates,may affect cells.

Prime Editing Pathogenic Transversion, Insertion, and Deletion Mutationsin Human Cells

The ability of PE3 to directly install or correct in human cellstransversion, small insertion, and small deletion mutations that causegenetic diseases, was tested. Sickle cell disease is most commonlycaused by an A·T-to-T·A transversion mutation in HBB, resulting in themutation of Glu6→Val in beta-globin. Treatment of hematopoietic stemcells ex vivo with Cas9 nuclease and a donor DNA template for HDR,followed by enrichment of edited cells, transplantation, and engraftmentis a promising potential strategy for the treatment of sickle-celldisease¹⁷⁰. However, this approach still generates many indel-containingbyproducts in addition to the correctly edited HBB allele¹⁷⁰⁻¹⁷¹. Whilebase editors generally produce far fewer indels, they cannot currentlymake the T·A-to-A·T transversion mutation needed to directly restore thenormal sequence of HBB.

PE3 was used to install the HBB E6V mutation in HEK293T cells with 44%efficiency and 4.8% indels (FIG. 43A. From the mixture of PE3-treatedcells, we isolated six HEK293T cell lines that are homozygous (triploid)for the HBB E6V allele (FIGS. 53A-53E), demonstrating the ability ofprime editing to generate human cell lines with pathogenic mutations. Tocorrect the HBB E6V allele to wild-type HBB, we treated homozygous HBBE6V HEK293T cells with PE3 and a PEgRNA programmed to directly revertthe HBB E6V mutation to wild-type HBB. In total, 14 PEgRNA designs weretested. After three days, DNA sequencing revealed that all 14 PEgRNAswhen combined with PE3 gave efficient correction of HBB E6V to wild-typeHBB (≥26% wild-type HBB without indels), and indel levels averaging2.8±0.70% (FIG. 50A). The best PEgRNA resulted in 52% correction of HBBE6V to wild-type with 2.4% indels (FIG. 43A). Introduction of a silentmutation that modifies the PAM recognized by the PEgRNA modestlyimproved editing efficiency and product purity, to 58% correction with1.4% indels (FIG. 43A). These results establish that prime editing caninstall and correct a pathogenic transversion point mutation in a humancell line with high efficiency and minimal byproducts.

Tay-Sachs disease is most often caused by a 4-bp insertion into the HEXAgene (HEXA 1278+TATC)¹³⁶. PE3 was used to install this 4-bp insertioninto HEK293T cells with 31% efficiency and 0.8% indels (FIG. 43B), andisolated two HEK293T cell lines that are homozygous for the HEXA1278+TATC allele (FIGS. 53A-53E). These cells were used to test 43PEgRNAs and three nicking sgRNAs with PE3 or PE3b systems for correctionof the pathogenic insertion in HEXA (FIG. 50B), either by perfectreversion to the wild-type allele or by a shifted 4-bp deletion thatdisrupts the PAM and installs a silent mutation. Nineteen of the 43PEgRNAs tested resulted in ≥20% editing. Perfect correction to wild-typeHEXA with PE3 or PE3b and the best PEgRNA proceeded with similar averageefficiencies (30% for PE3 vs. 33% for PE3b), but the PE3b system wasaccompanied by 5.3-fold fewer indel products (1.7% for PE3 vs. 0.32% forPE3b) (FIG. 43B and FIG. 50B). These findings demonstrate the ability ofprime editing to make precise small insertions and deletions thatinstall or correct a pathogenic allele in mammalian cells efficientlyand with a minimum of byproducts.

Finally, the installation of a protective SNP into PRNP, the geneencoding the human prion protein (PrP), was tested. PrP misfoldingcauses progressive and fatal neurodegenerative prion disease that canarise spontaneously, through inherited dominant mutations in the PRNPgene, or through exposure to misfolded PrP¹⁷². A naturally occurringPRNP G127V mutant allele confers resistance to prion disease inhumans¹³⁸ and mice¹⁷³. PE3 was used to install G127V into the human PRNPallele in HEK293T cells, which requires a G·C-to-T·A transversion. FourPEgRNAs and three nicking sgRNAs were evaluated with the PE3 system.After three days of exposure to the most effective PE3 and PEgRNA, DNAsequencing revealed 53±11% efficiency of installing the G127V mutationand indel levels of 1.7i0.7% (FIG. 43C). Taken together, these resultsestablish the ability of prime editing in human cells to install orcorrect transversion, insertion, or deletion mutations that cause orconfer resistance to disease efficiently, and with a minimum ofbyproducts.

Prime Editing in Various Human Cell Lines and Primary Mouse Neurons

Next, prime editing was tested for its ability to edit endogenous sitesin three additional human cell lines. In K562 (leukemic bone marrow)cells, PE3 was used to perform transversion edits in the HEK3, EMX1, andFANCF sites, as well as the 18-bp insertion of a 6×His tag in HEK3. Anaverage editing efficiency of 15-30% was observed for each of these fourPE3-mediated edits, with indels averaging 0.85-2.2% (FIG. 43A). In U2OS(osteosarcoma) cells, transversion mutations in HEK3 and FANCF wereinstalled, as well as a 3-bp insertion and 6×His tag insertion intoHEK3, with 7.9-22% editing efficiency that exceeded indel formation 10-to 76 fold(FIG. 43A). Finally, in HeLa (cervical cancer) cells, a 3-bpinsertion into HEK3 was performed, with 12% average efficiency and 1.3%indels (FIG. 43A). Collectively, these data indicate that multiple celllines beyond HEK293T cells support prime editing, although editingefficiencies vary by cell type and are generally less efficient than inHEK293T cells. Editing:indel ratios remained high in all tested humancell lines.

To determine if prime editing is possible in post-mitotic, terminallydifferentiated primary cells, primary cortical neurons harvested fromE18.5 mice were transduced with a dual split-PE3 lentiviral deliverysystem in which split-intein splicing²⁰³ reconstitutes PE2 protein fromN-terminal and C-terminal halves, each delivered from a separate virus.To restrict editing to post-mitotic neurons, the human synapsinpromoter, which is highly specific for mature neurons²⁰⁴, was used todrive expression of both PE2 protein components. GFP was fused through aself-cleaving P2A peptide²⁰⁵ to the N-terminal half of PE2. Nuclei fromneurons were isolated two weeks following dual viral transduction andwere sequenced directly, or sorted for GFP expression before sequencing.A 7.1±1.2% average prime editing to install a transversion at the DNMT1locus with 0.58±0.14% average indels in sorted nuclei (FIG. 43D wasobserved. Cas9 nuclease in the same split-intein dual lentivirus systemresulted in 31±5.5% indels among sorted cortical neuron nuclei (FIG.43D. These data indicate that post-mitotic, terminally differentiatedprimary cells can support prime editing, and thus establish that primeediting does not require cell replication. Prime editing compared withCas9-initiated HDR

The performance of PE3 was compared with that of optimizedCas9-initiated HDR^(128,125) in mitotic cell lines that support HDR¹²⁸.HEK293T, HeLa, K562 and U2OS cells were treated with Cas9 nuclease, asgRNA, and an ssDNA donor oligonucleotide template designed to install avariety of transversion and insertion edits (FIGS. 43E-43G, and FIGS.51A-51G). Cas9-initiated HDR in all cases successfully installed thedesired edit, but with far higher levels of byproducts (predominantlyindels), as expected from treatments that cause double-stranded breaks.Using PE3 in HEK293T cells, HBB E6V installation and correctionproceeded with 42% and 58% average editing efficiency with 2.6% and 1.4%average indels, respectively (FIG. 43E and FIG. 43G). In contrast, thesame edits with Cas9 nuclease and an HDR template resulted in 5.2% and6.7% average editing efficiency, with 79% and 51% average indelfrequency (FIG. 43E and FIG. 43G). Similarly, PE3 installed PRNP G127Vwith 53% efficiency and 1.7% indels, whereas Cas9-initiated HDRinstalled this mutation with 6.9% efficiency and 53% indels (FIG. 43Eand FIG. 43G). Thus, the ratio of editing:indels for HBB E6Vinstallation, HBB E6V correction, and PRNP G127V installation on averagewas 270-fold higher for PE3 than for Cas9-initiated HDR.

Comparisons between PE3 and HDR in human cell lines other than HEK293Tshowed similar results, although with lower PE3 editing efficiencies.For example, in K562 cells, PE3-mediated 3-bp insertion into HEK3proceeded with 25% efficiency and 2.8% indels, compared with 17% editingand 72% indels for Cas9-initiated HDR, a 40-fold editing:indel ratioadvantage favoring PE3 (FIGS. 43F-43G). In U2OS cells, PE3 performedthis 3-bp insertion with 22% efficiency and 2.2% indels, whileCas9-initiated HDR resulted in 15% editing with 74% indels, a 49-foldlower editing:indel ratio (FIGS. 43F-43G). In HeLa cells, PE3 made thisinsertion with 12% efficiency and 1.3% indels, versus 3.0% editing and69% indels for Cas9-initiated HDR, a 210-fold editing:indel ratiodifference (FIGS. 43F-43G). Collectively, these data indicated that HDRtypically results in similar or lower editing efficiencies and farhigher indels than PE3 in the four cell lines tested (FIGS. 51A-51G).

Discussion and Future Directions

The ability to insert DNA sequences with single-nucleotide precision isan especially enabling prime editing capability. For example, PE3 wasused to precisely insert into the HEK3 locus in HEK293T cells a His6 tag(18 bp, 65% average efficiency), a FLAG epitope tag (24 bp, 18% averageefficiency), and an extended LoxP site (44 bp, 23% average efficiency)that is the native substrate for Cre recombinase. Average indels rangedbetween 3.0% and 5.9% for these examples (FIG. 43H). Manybiotechnological, synthetic biology, and therapeutic applications areenvisioned to arise from the ability to efficiently and preciselyintroduce new DNA sequences into target sites of interest in livingcells.

Collectively, the prime editing experiments described herein installed18 insertions up to 44 bp, 22 deletions up to 80 bp, 113 point mutationsincluding 77 transversions, and 18 combination edits, across 12endogenous loci in the human and mouse genomes at locations ranging from3 bp upstream to 29 bp downstream of the start of a PAM without makingexplicit double-stranded DNA breaks. These results establish primeediting as a remarkably versatile genome editing method. Because theoverwhelming majority (85-99%) of insertions, deletions, indels, andduplications in ClinVar are ≤30 bp (FIGS. 52A-52D), in principle primeediting can correct up to ˜89% of the 75,122 currently known pathogenichuman genetic variants in ClinVar (transitions, transversions,insertions, deletions, indels, and duplications in FIG. 38A), withadditional potential to ameliorate diseases caused by copy number gainor loss.

Importantly, for any desired edit the flexibility of prime editingoffers many possible choices of PEgRNA-induced nick locations,sgRNA-induced second nick locations, PBS lengths, RT template lengths,and which strand to edit first, as demonstrated extensively herein. Thisflexibility, which contrasts with more limited options typicallyavailable for other precision genome editing methods^(124,142,154),allows editing efficiency, product purity, DNA specificity, or otherparameters to be optimized to suit the needs of a given application, asshown in FIGS. 50A-50B in which testing 14 and 43 PEgRNAs covering arange of prime editing strategies optimized correction of pathogenic HBBand HEXA alleles, respectively.

Much additional research is needed to further understand and improveprime editing. Additional modifications of prime editor systems may berequired to expand their compatibility to include other cell types, suchas post-mitotic cells. Interfacing prime editing with viral andnon-viral in vitro and in vivo delivery strategies is needed to fullyexplore the potential of prime editing to enable a wide range ofapplications including the study and treatment of genetic diseases. Byenabling highly precise targeted transitions, transversions, smallinsertions, and small deletions in the genomes of mammalian cellswithout requiring double-stranded breaks or HDR, however, prime editingprovides a new “search-and-replace” capability that substantiallyexpands the scope of genome editing.

Methods General Methods

DNA amplification was conducted by PCR using Phusion U Green MultiplexPCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity2× Master Mix (New England BioLabs) unless otherwise noted. DNAoligonucleotides, including Cy5-labeled DNA oligonucleotides, dCas9protein, and Cas9 H840A protein were obtained from Integrated DNATechnologies. Yeast reporter plasmids were derived from previouslydescribed plasmids64 and cloned by the Gibson assembly method. Allmammalian editor plasmids used herein were assembled using the USERcloning method as previously described¹⁷⁵. Plasmids expressing sgRNAswere constructed by ligation of annealed oligonucleotides intoBsmBI-digested acceptor vector. Plasmids expressing PEgRNAs wereconstructed by Gibson assembly or Golden Gate assembly using a customacceptor plasmid (see supplemental ‘Golden Gate assembly’ outline).Sequences of sgRNA and PEgRNA constructs used herein are listed inTables 2A-2C and Tables 3A-3R. All vectors for mammalian cellexperiments were purified using Plasmid Plus Midiprep kits (Qiagen) orPureYield plasmid miniprep kits (Promega), which include endotoxinremoval steps. All experiments using live animals were approved by theBroad Institute Institutional and Animal Care and Use Committees.Wild-type C57BL/6 mice were obtained from Charles River (#027).

In Vitro Biochemical Assays

PEgRNAs and sgRNAs were transcribed in vitro using the HiScribe T7 invitro transcription kit (New England Biolabs) from PCR-amplifiedtemplates containing a T7 promoter sequence. RNA was purified bydenaturing urea PAGE and quality-confirmed by an analytical gel prior touse. 5′-Cy5-labeled DNA duplex substrates were annealed using twooligonucleotides (Cy5-AVA024 and AVA025; 1:1.1 ratio) for the non-nickedsubstrate or three oligonucleotides (Cy5-AVA023, AVA025 and AVA026;1:1.1:1.1) for the pre-nicked substrate by heating to 95° C. for 3minutes followed by slowly cooling to room temperature (Tables 2A-2C).Cas9 cleavage and reverse transcription reactions were carried out in 1×cleavage buffer²⁰⁵ supplemented with dNTPs (20 mM HEPES-K, pH 7.5; 100mM KCl; 5% glycerol; 0.2 mM EDTA, pH 8.0; 3 mM MgCl2; 0.5 mM dNTP mix; 5mM DTT). dCas9 or Cas9 H840A (5 μM final) and the sgRNA or PEgRNA (5 μMfinal) were pre-incubated at room temperature in a 5 μL reaction mixturefor 10 minutes prior to the addition of duplex DNA substrate (400 nMfinal), followed by the addition of Superscript III reversetranscriptase (ThermoFisher Scientific), an undisclosed M-MLV RTvariant, when applicable. Reactions were carried out at 37° C. for 1hour, then diluted to a volume of 10 μL with water, treated with 0.2 μLof proteinase K solution (20 mg/mL, ThermoFisher Scientific), andincubated at room temperature for 30 minutes. Following heatinactivation at 95° C. for 10 minutes, reaction products were combinedwith 2x formamide gel loading buffer (90% formamide; 10% glycerol; 0.01%bromophenol blue), denatured at 95° C. for 5 minutes, and separated bydenaturing urea-PAGE gel (15% TBE-urea, 55° C., 200V). DNA products werevisualized by Cy5 fluorescence signal using a Typhoon FLA 7000biomolecular imager.

Electrophoretic mobility shift assays were carried out in Ix bindingbuffer (lx cleavage buffer+10 μg/mL heparin) using pre-incubateddCas9:sgRNA or dCas9:PEgRNA complexes (concentration range between 5 nMand 1 μM final) and Cy5-labeled duplex DNA (Cy5-AVA024 and AVA025; 20 nMfinal). After 15 minutes of incubation at 37° C., the samples wereanalyzed by native PAGE gel (10% TBE) and imaged for Cy5 fluorescence.

For DNA sequencing of reverse transcription products, fluorescent bandswere excised and purified from urea-PAGE gels, then 3′ tailed withterminal transferase (TdT; New England Biolabs) in the presence of dGTPor dATP according to the manufacturer's protocol. Tailed DNA productswere diluted 10-fold with binding buffer (40% saturated aqueousguanidinium chloride+60% isopropanol) and purified by QIAquick spincolumn (Qiagen), then used as templates for primer extension by Klenowfragment (New England Biolabs) using primer AVA134 (A-tailed products)or AVA135 (G-tailed products) (Tables 2A-2C). Extension were amplifiedby PCR for 10 cycles using primers AVA110 and AVA122, then sequencedwith AVA037 using the Sanger method (Tables 2A-2C).

Yeast Fluorescent Reporter Assays

Dual fluorescent reporter plasmids containing an in-frame stop codon, a+1 frameshift, or a −1 frameshift were subjected to 5′-extended PEgRNAor 3′-extended PEgRNA prime editing reactions in vitro as describedabove. Following incubation at 37° C. for 1 hour, the reactions werediluted with water and plasmid DNA was precipitated with 0.3 M sodiumacetate and 70% ethanol. Resuspended DNA was transformed into S.cerevisiae by electroporation as previously described⁶⁷ and plated onsynthetic complete media without leucine (SC(glucose), L-). GFP andmCherry fluorescence signals were visualized from colonies with theTyphoon FLA 7000 biomolecular imager.

General Mammalian Cell Culture Conditions

HEK293T (ATCC CRL-3216), U2OS (ATTC HTB-96), K562 (CCL-243), and HeLa(CCL-2) cells were purchased from ATCC and cultured and passaged inDulbecco's Modified Eagle's Medium (DMEM) plus GlutaMAX (ThermoFisherScientific), McCoy's 5A Medium (Gibco), RPMI Medium 1640 plus GlutaMAX(Gibco), or Eagle's Minimal Essential Medium (EMEM, ATCC), respectively,each supplemented with 10% (v/v) fetal bovine serum (Gibco, qualified)and 1× Penicillin Streptomycin (Corning). All cell types were incubated,maintained, and cultured at 37° C. with 5% CO₂. Cell lines wereauthenticated by their respective suppliers and tested negative formycoplasma.

HEK293T Tissue Culture Transfection Protocol and Genomic DNA Preparation

HEK293T cells grown were seeded on 48-well poly-D-lysine coated plates(Corning). 16 to 24 hours post-seeding, cells were transfected atapproximately 60% confluency with 1 μL of Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer's protocols and 750 ngof PE plasmid, 250 ng of PEgRNA plasmid, and 83 ng of sgRNA plasmid (forPE3 and PE3b). Unless otherwise stated, cells were cultured 3 daysfollowing transfection, after which the media was removed, the cellswere washed with 1×PBS solution (Thermo Fisher Scientific), and genomicDNA was extracted by the addition of 150 μL of freshly prepared lysisbuffer (10 mM Tris-HCl, pH 7.5; 0.05% SDS; 25 μg/mL Proteinase K(ThermoFisher Scientific)) directly into each well of the tissue cultureplate. The genomic DNA mixture was incubated at 37° C. for 1 to 2 hours,followed by an 80° C. enzyme inactivation step for 30 minutes. Primersused for mammalian cell genomic DNA amplification are listed in Table 4.For HDR experiments in HEK293T cells, 231 ng of nuclease-expressionplasmid, 69 ng of sgRNA expression plasmid, 50 ng (1.51 pmol) 100-ntssDNA donor template (PAGE-purified; Integrated DNA Technologies) waslipofected using 1.4 μL Lipofectamine 2000 (ThermoFisher) per well.Genomic DNA from all HDR experiments was purified using the AgencourtDNAdvance Kit (Beckman Coulter), according to the manufacturer'sprotocol.

High-Throughput DNA Sequencing of Genomic DNA Samples

Genomic sites of interest were amplified from genomic DNA samples andsequenced on an Illumina MiSeq as previously described with thefollowing modifications^(129,130). Briefly, amplification primerscontaining Illumina forward and reverse adapters (Table 4) were used fora first round of PCR (PCR 1) amplifying the genomic region of interest.25 μL PCR 1 reactions were performed with 0.5 μM of each forward andreverse primer, 1 μL of genomic DNA extract and 12.5 μL of Phusion UGreen Multiplex PCR Master Mix. PCR reactions were carried out asfollows: 98° C. for 2 minutes, then 30 cycles of [98° C. for 10 seconds,61° C. for 20 seconds, and 72° C. for 30 seconds], followed by a final72° C. extension for 2 minutes. Unique Illumina barcoding primer pairswere added to each sample in a secondary PCR reaction (PCR 2).Specifically, 25 μL of a given PCR 2 reaction contained 0.5 μM of eachunique forward and reverse illumina barcoding primer pair, 1 μL ofunpurified PCR 1 reaction mixture, and 12.5 μL of Phusion U GreenMultiplex PCR 2× Master Mix. The barcoding PCR 2 reactions were carriedout as follows: 98° C. for 2 minutes, then 12 cycles of [98° C. for 10seconds, 61° C. for 20 seconds, and 72° C. for 30 seconds], followed bya final 72° C. extension for 2 minutes. PCR products were evaluatedanalytically by electrophoresis in a 1.5% agarose gel. PCR 2 products(pooled by common amplicons) were purified by electrophoresis with a1.5% agarose gel using a QIAquick Gel Extraction Kit (Qiagen), elutingwith 40 μL of water. DNA concentration was measured by fluorometricquantification (Qubit, ThermoFisher Scientific) or qPCR (KAPA LibraryQuantification Kit-Illumina, KAPA Biosystems) and sequenced on anIllumina MiSeq instrument according to the manufacturer's protocols.

Sequencing reads were demultiplexed using MiSeq Reporter (Illumina).Alignment of amplicon sequences to a reference sequence was performedusing CRISPResso2¹⁷⁸. For quantification of point mutation editing,CRISPResso2 was run in standard mode with “discard_indel_reads” on.Editing efficiency was calculated as: (frequency of specified pointmutation in non-discarded reads)×(#of non-discarded reads)÷total reads.For insertion or deletion edits, CRISPResso2 was run in HDR mode usingthe desired allele as the expected allele (e flag), and with“discard_indel_reads” ON. Editing yield was calculated as the number ofHDR aligned reads divided by total reads. For all edits, indel yieldswere calculated as the number of discarded reads divided by total reads.

Nucleofection of U2OS, K562, and HeLa Cells

Nucleofection was performed in all experiments using K562, HeLa, andU20S cells. For PE conditions in these cell types, 800 ng primeeditor-expression plasmid, 200 ng PEgRNA-expression plasmid, and 83 ngnicking plasmid was nucleofected in a final volume of 20 μL in a 16-wellnucleocuvette strip (Lonza). For HDR conditions in these three celltypes, 350 ng nuclease-expression plasmid, 150 ng sgRNA-expressionplasmid and 200 pmol (6.6 μg) 100-nt ssDNA donor template(PAGE-purified; Integrated DNA Technologies) was nucleofected in a finalvolume of 20 μL per sample in a 16-well Nucleocuvette strip (Lonza).K562 cells were nucleofected using the SF Cell Line 4D-Nucleofector XKit (Lonza) with 5×10⁵ cells per sample (program FF-120), according tothe manufacturers protocol. U2OS cells were nucleofected using the SECell Line 4D-Nucleofector X Kit (Lonza) with 3-4×10⁵ cells per sample(program DN-100), according to the manufacturers protocol. HeLa cellswere nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza)with 2×10⁵ cells per sample (program CN-114), according to themanufacturers protocol. Cells were harvested 72 hours afternucleofection for genomic DNA extraction.

Genomic DNA Extraction for HDR Experiments

Genomic DNA from all HDR comparison experiments in HEK293T, HEK293T HBBE6V, K562, U20S, and HeLa cells was purified using the AgencourtDNAdvance Kit (Beckman Coulter), according to the manufacturer'sprotocol.

Comparison Between PE2, PE3, BE2, BE4max, ABEdmax, and ABEmax

HEK293T cells were seeded on 48-well poly-D-lysine coated plates(Corning). After 16 to 24 hours, cells were transfected at approximately60% confluency. For base editing with CBE or ABE constructs, cells weretransfected with 750 ng of base editor plasmid, 250 ng of sgRNAexpression plasmid, and 1 μL of Lipofectamine 2000 (Thermo FisherScientific). PE transfections were performed as described above. GenomicDNA extraction for PE and BE was performed as described above.

Determination of PE3 Activity at Known Cas9 Off-Target Sites

To evaluate PE3 off-target editing activity at known Cas9 off-targetsites, genomic DNA extracted from HEK293T cells 3 days aftertransfection with PE3 was used as template for PCR amplification of 16previously reported Cas9 off-target genomic sites^(118,159) (the topfour off-target sites each for the HEK3, EMX1, FANCF, and HEK4 spacers;primer sequences are listed in Table 4). These genomic DNA samples wereidentical to those used for quantifying on-target PE3 editing activitiesshown in FIGS. 41A-41K; PEgRNA and nicking sgRNA sequences are listed inTables 3A-3R. Following PCR amplification of off-target sites, ampliconswere sequenced on the Illumina MiSeq platform as described above (HTSanalysis). For determining Cas9 nuclease, Cas9 H840A nickase, dCas9, andPE2-dRT on-target and off-target editing activity, HEK293T cells weretransfected with 750 ng of editor plasmid (Cas9 nuclease, Cas9 H840Anickase, dCas9, or PE2-dRT), 250 ng of PEgRNA or sgRNA plasmid, and 1 μLof Lipofectamine 2000. Genomic DNA was isolated from cells 3 days aftertransfection as described above. On-target and off-target genomic lociwere amplified by PCR using primer sequences in Table 4 and sequenced onan Illumina MiSeq.

HTS data analysis was performed using CRISPResso2¹⁷⁸. The editingefficiencies of Cas9 nuclease, Cas9 H840A nickase, and dCas9 werequantified as the percent of total sequencing reads containing indels.For quantification of PE3 and PE3-dRT off-targets, aligned sequencingreads were examined for point mutations, insertions, or deletions thatwere consistent with the anticipated product of PEgRNA reversetranscription initiated at the Cas9 nick site. Single nucleotidevariations occurring at <0.1% overall frequency among total reads withina sample were excluded from analysis. For reads containing singlenucleotide variations that both occurred at frequencies ≥0.1% and werepartially consistent with the PEgRNA-encoded edit, t-tests (unpaired,one-tailed, α=0.5) were used to determine if the variants occurred atsignificantly higher levels compared to samples treated with PEgRNAsthat contained the same spacer but encoded different edits. To avoiddifferences in sequencing errors, comparisons were made between samplesthat were sequenced simultaneously within the same MiSeq run. Variantsthat did not meet the criteria of p-value >0.05 were excluded.Off-target PE3 editing activity was then calculated as the percentage oftotal sequencing reads that met the above criteria.

Generation of a HEK293T Cell Line Containing the HBB E6V Mutation UsingCas9-Initiated HDR

HEK293T cells were seeded in a 48-well plate and transfected atapproximately 60% confluency with 1.5 μL of Lipofectamine 2000, 300 ngof Cas9 D10A nickase plasmid, 100 ng of sgRNA plasmid, and 200 ng of100-mer ssDNA donor template (Table 5). Three days after transfection,media was exchanged for fresh media. Four days after transfection, cellswere dissociated using 30 μL of TrypLE solution and suspended in 1.5 mLof media. Single cells were isolated into individual wells of two96-well plates by fluorescence-activated cell sorting (FACS)(Beckman-Coulter Astrios). See FIGS. 53A-53B for representative FACSsorting examples. Cells were expanded for 14 days prior to genomic DNAsequencing as described above. Of the isolated clonal populations, nonewas found to be homozygous for the HBB E6V mutation, so a second roundof editing by lipofection, sorting, and outgrowth was repeated in apartially edited cell line to yield a cell line homozygous for the E6Vallele.

Generation of a HEK293T Cell Line Containing the HBB E6V Mutation UsingPE3

2.5×104 HEK293T cells grown in the absence of antibiotic were seeded on48-well poly-D-lysine coated plates (Corning). 16 to 24 hourspost-seeding, cells were transfected at approximately 70% confluencywith 1 μL of Lipofectamine 2000 (Thermo Fisher Scientific) according tothe manufacturer's protocols and 750 ng of PE2-P2A-GFP plasmid, 250 ngof PEgRNA plasmid, and 83 ng of sgRNA plasmid. After 3 days posttransfection, cells were washed with phosphate-buffered saline (Gibco)and dissociated using TrypLE Express (Gibco). Cells were then dilutedwith DMEM plus GlutaMax (Thermo Fisher Scientific) supplemented with 10%(v/v) FBS (Gibco) and passed through a 35-μm cell strainer (Corning)prior to sorting. Flow cytometry was carried out on a LE-MA900 cellsorter (Sony). Cells were treated with 3 nM DAPI (BioLegend) 15 minutesprior to sorting. After gating for doublet exclusion, singleDAPI-negative cells with GFP fluorescence above that of a GFP-negativecontrol cell population were sorted into 96-well flat-bottom cellculture plates (Corning) filled with pre-chilled DMEM with GlutaMaxsupplemented with 10% FBS. See FIGS. 53A-53B for representative FACSsorting examples. Cells were cultured for 10 days prior to genomic DNAextraction and characterization by HTS, as described above. A total ofsix clonal cell lines were identified that are homozygous for the E6Vmutation in HBB.

Generation of a HEK293T Cell Line Containing the HEXA 1278+TATCInsertion Using PE3

HEK293T cells containing the HEXA 1278+TATC allele were generatedfollowing the protocol described above for creation of the HBB E6V cellline; PEgRNA and sgRNA sequences are listed in Tables 2A-2C under theFIGS. 43A-43H subheading. After transfection and sorting, cells werecultured for 10 days prior to genomic DNA extraction andcharacterization by HTS, as described above. Two heterozygous cell lineswere isolated that contained 50% HEXA 1278+TATC alleles, and twohomozygous cell lines containing 100% HEXA 1278+TATC alleles wererecovered.

Cell Viability Assays

HEK293T cells were seeded in 48-well plates and transfected atapproximately 70% confluency with 750 ng of editor plasmid (PE3, PE3R110S K103L, Cas9 H840A nickase, or dCas9), 250 ng of HEK3-targetingPEgRNA plasmid, and 1 μL of Lipofectamine 2000, as described above. Cellviability was measured every 24 hours post-transfection for 3 days usingthe CellTiter-Glo 2.0 assay (Promega) according to the manufacturer'sprotocol. Luminescence was measured in 96-well flat-bottomed polystyrenemicroplates (Corning) using a M1000 Pro microplate reader (Tecan) with a1-second integration time.

Lentivirus Production

Lentivirus was produced as previously described²⁰⁶. T-75 flasks ofrapidly dividing HEK293T cells (ATCC; Manassas, VA, USA) weretransfected with lentivirus production helper plasmids pVSV-G and psPAX2in combination with modified lentiCRISPR_v2 genomes carryingintein-split PE2 editor using FuGENE HD (Promega, Madison, WI, USA)according to the manufacturer's directions. Four split-intein editorconstructs were designed: 1) a viral genome encoding a U6-PEgRNAexpression cassette and the N-terminal portion (1-573) of Cas9 H840Anickase fused to the Npu N-intein, a self-cleaving P2A peptide, andGFP-KASH; 2) a viral genome encoding the Npu C-intein fused to theC-terminal remainder of PE2; 3) a viral genome encoding the Npu C-inteinfused to the C-terminal remainder of Cas9 for the Cas9 control; and 4) anicking sgRNA for DNMT1. The split-intein mediates trans splicing tojoin the two halves of PE2 or Cas9, while the P2A GFP-KASH enablesco-translational production of a nuclear membrane-localized GFP. After48 hours, supernatant was collected, centrifuged at 500 g for 5 minutesto remove cellular debris, and filtered using a 0.45 μm filter. Filteredsupernatant was concentrated using the PEG-it Virus PrecipitationSolution (System Biosciences, Palo Alto, CA, USA) according to themanufacturer's directions. The resulting pellet was resuspended inOpti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) using 1% of theoriginal media volume. Resuspended pellet was flash-frozen and stored at−80° C. until use.

Mouse Primary Cortical Neuron Dissection and Culture

E18.5 dissociated cortical cultures were harvested from timed-pregnantC57BL/6 mice (Charles River). Embryos were harvested from pregnant miceafter euthanasia by CO2 followed by decapitation. Cortical caps weredissected in ice-cold Hibernate-E supplemented withpenicillin/streptomycin (Life Technologies). Following a rinse withice-cold Hibernate-E, tissue was digested at 37° C. for 8 minutes inpapain/DNase (Worthington/Sigma). Tissue was triturated in NBActiv4(BrainBits) supplemented with DNase. Cells were counted and plated in24-well plates at 100,000 cells per well. Half of the media was changedtwice per week.

Prime Editing in Primary Neurons and Nuclei Isolation

At DIV 1, 15 μL of lentivirus was added at 10:10:1 ratio ofN-terminal:C-terminal:nicking sgRNA. At DIV 14, neuronal nuclei wereisolated using the EZ-PREP buffer (Sigma D8938) following themanufacturer's protocol. All steps were performed on ice or at 4° C.Media was removed from dissociated cultures, and cultures were washedwith ice-cold PBS. PBS was aspirated and replaced with 200 μL EZ-PREPsolution. Following a 5-minute incubation on ice, EZ-PREP was pipettedacross the surface of the well to dislodge remaining cells. The samplewas centrifuged at 500 g for 5 minutes, and the supernatant removed.Samples were washed with 200 μL EZ-PREP and centrifuged again at 500 gfor 5 minutes. Samples were resuspended with gentle pipetting in 200 μLice-cold Nuclei Suspension Buffer (NSB) consisting of 100 μg/mL BSA and3.33 μM Vybrant DyeCycle Ruby (Thermo Fisher) in 1×PBS, then centrifugedat 500 g for 5 minutes. The supernatant was removed and nuclei wereresuspended in 100 μL NSB and sorted into 100 μL Agencourt DNAdvancelysis buffer using a MoFlo Astrios (Beckman Coulter) at the BroadInstitute flow cytometry facility. Genomic DNA was purified according tothe manufacturer's Agencourt DNAdvance instructions.

RNA-Sequencing and Data Analysis

HEK293T cells were co-transfected with PRNP-targeting or HEXA-targetingPEgRNAs and PE2, PE2-dRT, or Cas9 H840A nickase. 72 hours followingtransfection, total RNA was harvested from cells using TRIzol reagent(Thermo Fisher) and purified with RNeasy Mini kit (Qiagen) includingon-column DNaseI treatment. Ribosomes were depleted from total RNA usingthe rRNA removal protocol of the TruSeq Stranded Total RNA library prepkit (Illumina) and subsequently washed with RNAClean XP beads (BeckmanCoulter). Sequencing libraries were prepared using ribo-depleted RNA ona SMARTer PrepX Apollo NGS library prep system (Takara) following themanufacturer's protocol. Resulting libraries were visualized on a 2200TapeStation (Agilent Technologies), normalized using a Qubit dsDNA HSassay (Thermo Fisher), and sequenced on a NextSeq 550 using high outputv2 flow cell (Illumina) as 75-bp paired-end reads. Fastq files weregenerated with bcl2fastq2 version 2.20 and trimmed using TrimGaloreversion 0.6.2 (github.com/FelixKrueger/TrimGalore) to remove low-qualitybases, unpaired sequences, and adaptor sequences. Trimmed reads werealigned to a Homo sapiens genome assembly GRCh¹⁴⁸ with a custom Cas9H840A gene entry using RSEM version 1.3.1²⁰⁷. The limma-voom²⁰⁸ packagewas used to normalize gene expression levels and perform differentialexpression analysis with batch effect correction. Differentiallyexpressed genes were called with FDR-corrected p-value <0.05 andfold-change >2 cutoffs, and results were visualized in R.

ClinVar Analysis

The ClinVar variant summary was downloaded from NCBI (accessed Jul. 15,2019), and the information contained therein was used for all downstreamanalysis. The list of all reported variants was filtered by allele ID inorder to remove duplicates and by clinical significance in order torestrict the analysis to pathogenic variants. The list of pathogenicvariants was filtered sequentially by variant type in order to calculatethe fraction of pathogenic variants that are insertions, deletions, etc.Single nucleotide variants (SNVs) were separated into two categories(transitions and transversions) based on the reported reference andalternate alleles. SNVs that did not report reference or alternatealleles were excluded from the analysis.

The lengths of reported insertions, deletions, and duplications werecalculated using reference/alternate alleles, variant start/stoppositions, or appropriate identifying information in the variant name.Variants that did not report any of the above information were excludedfrom the analysis. The lengths of reported indels (single variants thatinclude both insertions and deletions relative to the reference genome)were calculated by determining the number of mismatches or gaps in thebest pairwise alignment between the reference and alternate alleles.Frequency distributions of variant lengths were calculated usingGraphPad Prism 8.

Data Availability

High-throughput sequencing data are deposited to the NCBI Sequence ReadArchive database. Plasmids encoding PE1, PE2/PE3, and PEgRNA expressionvectors will be available from Addgene.

Code Availability

The script used to quantify PEgRNA scaffold insertion is provided inFIGS. 60A-60B.

Supplemental Information: Tables and Sequences

TABLE 1 Activities of prime editors, Cas9 nuclease, Cas9 H840A nickase,and PE2-dRT at HEK3, HEK4, EMX1, and FANCF on-target and off-targetsites. PE2/PE3 editing is shown as % prime editing alongside % indels(in parentheses). % indels are shown for Cas9, Cas9 H840A nickase(nCas9), and PE2-dRT at the top four previously characterized off-target sites^(179,180). sgRNA and PEgRNA sequences can be found inTables 3A-3R, under the FIGS. 42A-42H heading. All values are theaverage of three independent biological replicates. PE pegRNA HEK3 (PE3)HEK4 (PE2) EMX1 (PE3) FANCF (PE3) Site . . . 1 2 3 4 . . . 1 2 3 4 . . .1 2 3 4 . . . 1 2 3 4 On-target 44.2 61.2 40.4 48.4 18.2 14.4 9.8 7.928.8 14.1 35.7 15.4 56.8 32.4 42.8 47.6 (11.9) (8.8) (16.5) (3.3) (0.9)(1.8) (2.0) (2.2) (3.5) (2.4) (3.3) (2.9) (9.3) (16.7) (13.6) (12.9)Off-target 1 <0.1 <0.1 <0.1 <0.1 <0.1 0.4 <0.1 0.4 <0.1 <0.1 <0.1 <0.1<0.1 0.6 <0.1 <0.1 (<0.1) (<0.1) (<0.1) (<0.1) (<0.1) (<0.1) (<0.1)(<0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) Off-target 2 <0.1<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1<0.1 (<0.1) (<0.1) (<0.1) (<0.1) (0.1) (0.1) (0.1) (0.1) (<0.1) (0.1)(0.1) (0.1) (<0.1) (<0.1) (0.1) (<0.1) Off-target 3 <0.1 <0.1 <0.1 <0.10.2 6.8 19.2 7.9 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 (<0.1) (<0.1)(<0.1) (<0.1) (0.5) (1.9) (0.5) (8.5) (0.8) (0.3) (0.3) (0.3) (<0.1)(<0.1) (<0.1) (<0.1) Off-target 4 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 (<0.1) (<0.1) (<0.1) (<0.1)(<0.1) (<0.1) (0.2) (<0.1) (0.1) (0.1) (0.2) (0.1) (<0.1) (<0.1) (<0.1)(<0.1) Cas9 pegRNA HEK3 HEK4 EMX1 FANCF sgRNA 1 2 3 4 sgRNA 1 2 3 4sgRNA 1 2 3 4 sgRNA 1 2 3 4 On-target 91.8 87.5 89.2 89.1 86.8 71.8 68.672.8 72.8 70.9 85.6 79.7 70.6 76.6 76.0 78.7 55.9 58.3 51.8 52.0Off-target 1 17.2 1.9 5.5 5.2 1.8 54.2 39.5 48.4 49.7 49.2 81.1 63.548.1 53.0 59.6 12.6 1.9 1.9 1.7 1.7 Off-target 2 38.0 6.5 12.6 11.8 4.742.5 19.5 29.4 27.3 30.3 58.3 12.0 6.0 8.2 12.9 1.1 0.2 0.2 0.2 0.1Off-target 3 8.8 0.6 1.7 1.5 0.5 98.1 96.9 97.3 97.6 97.5 14.8 4.2 3.13.6 4.8 2.4 0.2 <0.1 0.2 0.2 Off-target 4 0.3 <0.1 <0.1 0.1 <0.1 45.316.9 28.0 27.5 29.7 39.5 1.3 0.9 0.6 1.3 1.0 0.2 0.2 0.2 0.2 nCas9pegRNA HEK3 HEK4 EMX1 FANCF Site . . . 1 2 3 4 . . . 1 2 3 4 . . . 1 2 34 . . . 1 2 3 4 Off-target 1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.10.1 <0.1 0.1 <0.1 <0.1 <0.1 <0.1 Off-target 2 <0.1 <0.1 <0.1 <0.1 <0.1<0.1 <0.1 <0.1 0.3 0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 Off-target 3 <0.1<0.1 <0.1 <0.1 0.3 0.5 0.7 0.7 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1Off-target 4 <0.1 <0.1 <0.1 <0.1 <0.1 0.1 <0.1 <0.1 1.5 0.1 0.1 0.4 <0.1<0.1 <0.1 <0.1 PE2-dRT pegRNA HEK3 HEK4 EMX1 FANCF Site . . . 1 2 3 4 .. . 1 2 3 4 . . . 1 2 3 4 . . . 1 2 3 4 Off-target 1 <0.1 <0.1 <0.1 <0.10.1 0.2 <0.1 <0.1 0.1 0.1 0.1 0.1 <0.1 <0.1 <0.1 <0.1 Off-target 2 <0.1<0.1 <0.1 <0.1 <0.1 0.1 <0.1 <0.1 <0.1 0.3 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1Off-target 3 <0.1 <0.1 <0.1 <0.1 <0.1 0.1 1.4 0.9 <0.1 <0.1 <0.1 <0.1<0.1 <0.1 <0.1 <0.1 Off-target 4 <0.1 <0.1 <0.1 <0.1 0.1 0.1 0.1 0.2 0.90.1 0.1 0.2 <0.1 <0.1 <0.1 <0.1

Tables 2A-2C: Sequences of DNA oligonucleotides, PEgRNAs, and sgRNAsused for in vitro experiments.

TABLE 2A DNA oligonucleotides OLIGONUCLEOTIDE SEQUENCE AVA0235CY5-CCTGGGTCAATCCTTGGGGCCCAGACTGAGCACG (SEQ ID NO: 374) AVA024 5CY5-CCTGGGTCAATCCTTGGGGCCCAGACTGAGCACGTGATGGCAGAGG AAAGG (SEQ ID NO: 375)AVA025 5PHOS- CCTTTCCTCTGCCATCACGTGCTCAGTCTGGGCCCCAAGGATTGACCCAGG (SEQ ID NO: 376) AVA026 5PHOS-TGATGGCAGAGGAAAGG (SEQ ID NO: 377)AVA037 GCAGGCTTTAAAGGAACCAATTC (SEQ ID NO: 378) AVA110GCAGGCTTTAAAGGAACCAATTCCCTGGGTCAATCCTTGGGGC (SEQ ID NO: 379) AVA122CTCTGGAGGATCTAGCGGAG (SEQ ID NO: 380) AVA134CTCTGGAGGATCTAGCGGAGTTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 381) AVA135CTCTGGAGGATCTAGCGGAGCCCCCCCCCCCCCC (SEQ ID NO: 382)

TABLE 2B 5′-extended PEgRNAs RT LINKER PBS TEMPLATE SPACER 5′ EXTENSIONLENGTH LENGTH LENGTH PEGRNA SEQUENCE SEQUENCE (NT) (NT) (NT) PEGRNA 1GGCCCAGACTG GGCTAACCGTGCCATT 15 5 7 AGCACGTGA TGATCAGGTCA (SEQ(SEQ ID NO: 383) ID NO: 429) PEGRNA 2 GGCCCAGACTG GGCTAACCGTGCAAA 15 5 7AGCACGTGA TTAACAAACTAA (SEQ (SEQ ID NO: 384) ID NO: 430) PEGRNA 3GGCCCAGACTG GGCCATCTCGTGCAA 15 5 8 AGCACGTGA ATTAACAAACTAA(SEQ ID NO: 385) (SEQ ID NO: 431) PEGRNA 4 GGCCCAGACTG GGTCCTCTGCCATCTC15 5 15 AGCACGTGA GTGCAAATTAACAAA (SEQ ID NO: 386) CTAA (SEQ ID NO: 432)PEGRNA 5 GGCCCAGACTG GGCTTCCTTTCCTCTG 15 5 22 AGCACGTGA CCATCTCGTGCAAATT(SEQ ID NO: 387) AACAAACTAA (SEQ ID NO: 433) 5′- GGCCCAGACTGGGCTAACCGTGCCATT 15 5 7 PEGRNA_RT_7_A AGCACGTGA TGATCAGGTCA (SEQ(SEQ ID NO: 388) ID NO: 434) 5′- GGCCCAGACTG GGCTAACCGTGCAAA 15 5 7PEGRNA_RT_7_B AGCACGTGA TTAACAAACTAA (SEQ (SEQ ID NO: 389) ID NO: 435)5′- GGCCCAGACTG GGCCATCTCGTGCAA 15 5 8 PEGRNA_RT_8 AGCACGTGAATTAACAAACTAA (SEQ ID NO: 390) (SEQ ID NO: 436) 5′- GGCCCAGACTGGGTCCTCTGCCATCTC 15 5 15 PEGRNA_RT_15 AGCACGTGA GTGCAAATTAACAAA(SEQ ID NO: 391) CTAA (SEQ ID NO: 437) 5′- GGCCCAGACTG GGCTTCCTTTCCTCTG15 5 22 PEGRNA_RT_22 AGCACGTGA CCATCTCGTGCAAATT (SEQ ID NO: 392)AACAAACTAA (SEQ ID NO: 438)

TABLE 2C 3′-extended PEgRNAs RT PBS TEMPLATE 3′ EXTENSION LENGTH LENGTHPEGRNA SPACER SEQUENCE SEQUENCE (NT) (NT) 3′-PEGRNA_10 GGCCCAGACTGAGTCTGCCATCTCG 7 10 CACGTGA(SEQ ID TGCTC (SEQ ID NO: 506) NO: 439) 3′-GGCCCAGACTGAG TCTGCCATCTCG 7 10 PEGRNA_YEAST_TTOA CACGTGA (SEQ IDTGCTC (SEQ ID NO: 507) NO: 440) 3′- GGCCCAGACTGAG TCTGCCATCATC 7 11PEGRNA_YEAST_+1AINS CACGTGA (SEQ ID GTGCTC (SEQ ID NO: 508) NO: 441) 3′-GGCCCAGACTGAG TCTGCCATCCGT 7 9 PEGRNA_YEAST_+1TDEL CACGTGA (SEQ IDGCTC (SEQ ID NO: 509) NO: 442)

Tables 3A-3R: Sequences of PEgRNAs and sgRNAs used in mammalian cellexperiments. All sequences are shown in 5′ to 3′ orientation. Toconstruct PEgRNAs, spacer sequences listed below were added to the 5′end of the sgRNA scaffold and the 3′ extensions listed below containingthe primer binding site and RT template were added to the 3′ end of thesgRNA scaffold. The sgRNA scaffold sequence is

(SEQ ID NO: 131) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC.

TABLE 3A FIGS. 39A-39D PEgRNA RT SPACER SEQUENCE PBS TEMPLATE(SEQ ID NOS: 3′ EXTENSION (SEQ ID LENGTH LENGTH PEGRNA 2890-2996)NOS: 2997-3103) (NT) (NT) HEK3_2B- GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 8 10C_8 CACGTGA HEK3_2B- GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 9 10 C_9 CACGTGA GHEK3_2B- GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 10 10 C_10 CACGTGA GT HEK3_2B-GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 11 10 C_11 CACGTGA GTC HEK3_2B-GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 12 10 C_12 CACGTGA GTCT HEK3_2B-GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 13 10 C_13 CACGTGA GTCTG HEK3_2B-GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 14 10 C_14 CACGTGA GTCTGG HEK3_2B-GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 15 10 C_15 CACGTGA GTCTGGG HEK3_2C_16GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 16 10 CACGTGA GTCTGGGC HEK3_2C_17GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 17 10 CACGTGA GTCTGGGCC EMX1_2C_9GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 9 13 AGAAGAA CTGC EMX1_2C_10GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 10 13 AGAAGAA CTGCT EMX1_2C_11GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 11 13 AGAAGAA CTGCTC EMX1_2C_12GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 12 13 AGAAGAA CTGCTCG EMX1_2C_13GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 13 13 AGAAGAA CTGCTCGG EMX1_2C_14GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 14 13 AGAAGAA CTGCTCGGA EMX1_2C_15GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 15 13 AGAAGAA CTGCTCGGAC EMX1_2C_16GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 16 13 AGAAGAA CTGCTCGGACT EMX1_2C_17GAGTCCGAGCAGA ATGGGAGCACTTCTTCTT 17 13 AGAAGAA CTGCTCGGACTC FANCF_2C_8GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 8 17 AGCACC GCTGCAGA FANCF_2C_9GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 9 17 AGCACC GCTGCAGAA FANCF_2C_10GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 10 17 AGCACC GCTGCAGAAG FANCF_2C_11GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 11 17 AGCACC GCTGCAGAAGG FANCF_2C_12GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 12 17 AGCACC GCTGCAGAAGGG FANCF_2C_13GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 13 17 AGCACC GCTGCAGAAGGGA FANCF_2C_14GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 14 17 AGCACC GCTGCAGAAGGGAT FANCF_2C_15GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 15 17 AGCACC GCTGCAGAAGGGATTFANCF_2C_16 GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 16 17 AGCACCGCTGCAGAAGGGATTC FANCF_2C_17 GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 17 17AGCACC GCTGCAGAAGGGATTCC RNF2_2C_9 GTCATCTTAGTCAT GAACACCTCATGTAATG 9 11TACCTG ACT RNF2_2C_10 GTCATCTTAGTCAT GAACACCTCATGTAATG 10 11 TACCTG ACTARNF2_2C_11 GTCATCTTAGTCAT GAACACCTCATGTAATG 11 11 TACCTG ACTAARNF2_2C_12 GTCATCTTAGTCAT GAACACCTCATGTAATG 12 11 TACCTG ACTAAGRNF2_2C_13 GTCATCTTAGTCAT GAACACCTCATGTAATG 13 11 TACCTG ACTAAGARNF2_2C_14 GTCATCTTAGTCAT GAACACCTCATGTAATG 14 11 TACCTG ACTAAGATRNF2_2C_15 GTCATCTTAGTCAT GAACACCTCATGTAATG 15 11 TACCTG ACTAAGATGRNF2_2C_16 GTCATCTTAGTCAT GAACACCTCATGTAATG 16 11 TACCTG ACTAAGATGARNF2_2C_17 GTCATCTTAGTCAT GAACACCTCATGTAATG 17 11 TACCTG ACTAAGATGACHEK4_2C_7 GGCACTGCGGCTG GCTTTAACCCCAACCTC 7 13 GAGGTGG CAG HEK4_2C_8GGCACTGCGGCTG GCTTTAACCCCAACCTC 8 13 GAGGTGG CAGC HEK4_2C_9GGCACTGCGGCTG GCTTTAACCCCAACCTC 9 13 GAGGTGG CAGCC HEK4_2C_10GGCACTGCGGCTG GCTTTAACCCCAACCTC 10 13 GAGGTGG CAGCCG HEK4_2C_11GGCACTGCGGCTG GCTTTAACCCCAACCTC 11 13 GAGGTGG CAGCCGC HEK4_2C_12GGCACTGCGGCTG GCTTTAACCCCAACCTC 12 13 GAGGTGG CAGCCGCA HEK4_2C_13GGCACTGCGGCTG GCTTTAACCCCAACCTC 13 13 GAGGTGG CAGCCGCAG HEK4_2C_14GGCACTGCGGCTG GCTTTAACCCCAACCTC 14 13 GAGGTGG CAGCCGCAGT HEK4_2C_15GGCACTGCGGCTG GCTTTAACCCCAACCTC 15 13 GAGGTGG CAGCCGCAGTG HEK3_2C_1TDELGGCCCAGACTGAG TCTGCCATCCGTGCTCAG 13 10 CACGTGA TCTG HEK3_2C_1AINSGGCCCAGACTGAG TCTGCCATCATCGTGCTC 13 10 CACGTGA AGTCTG HEK3_2C_1CTTINSGGCCCAGACTGAG TCTGCCATCAAAGCGTG 13 10 CACGTGA CTCAGTCTG HEK3_2D_10GGCCCAGACTGAG TCTGCCATCTCGTGCTCA 13 10 CACGTGA GTCTG HEK3_2D_11GGCCCAGACTGAG CTCTGCCATCTCGTGCTC 13 11 CACGTGA AGTCTG HEK3_2D_12GGCCCAGACTGAG CCTCTGCCATCTCGTGCT 13 12 CACGTGA CAGTCTG HEK3_2D_13GGCCCAGACTGAG TCCTCTGCCATCTCGTGC 13 13 CACGTGA TCAGTCTG HEK3_2D_14GGCCCAGACTGAG TTCCTCTGCCATCTCGTG 13 14 CACGTGA CTCAGTCTG HEK3_2D_15GGCCCAGACTGAG TTTCCTCTGCCATCTCGT 13 15 CACGTGA GCTCAGTCTG HEK3_2D_16GGCCCAGACTGAG CTTTCCTCTGCCATCTCG 13 16 CACGTGA TGCTCAGTCTG HEK3_2D_17GGCCCAGACTGAG CCTTTCCTCTGCCATCTC 13 17 CACGTGA GTGCTCAGTCTG HEK3_2D_18GGCCCAGACTGAG TCCTTTCCTCTGCCATCT 13 18 CACGTGA CGTGCTCAGTCTG HEK3_2D_19GGCCCAGACTGAG TTCCTTTCCTCTGCCATC 13 19 CACGTGA TCGTGCTCAGTCTG HEK3_2D_20GGCCCAGACTGAG CTTCCTTTCCTCTGCCAT 13 20 CACGTGA CTCGTGCTCAGTCTGEMX1_2D_10 GAGTCCGAGCAGA GGAGCCCTTGTTCTTCTG 13 10 AGAAGAA CTCGGEMX1_2D_11 GAGTCCGAGCAGA GGGAGCCCTTGTTCTTCT 13 11 AGAAGAA GCTCGGEMX1_2D_12 GAGTCCGAGCAGA TGGGAGCCCTTGTTCTTC 13 12 AGAAGAA TGCTCGGEMX1_2D_13 GAGTCCGAGCAGA ATGGGAGCCCTTGTTCTT 13 13 AGAAGAA CTGCTCGGEMX1_2D_14 GAGTCCGAGCAGA GATGGGAGCCCTTGTTC 13 14 AGAAGAA TTCTGCTCGGEMX1_2D_15 GAGTCCGAGCAGA TGATGGGAGCCCTTGTT 13 15 AGAAGAA CTTCTGCTCGGEMX1_2D_16 GAGTCCGAGCAGA GTGATGGGAGCCCTTGT 13 16 AGAAGAA TCTTCTGCTCGGEMX1_2D_17 GAGTCCGAGCAGA TGTGATGGGAGCCCTTG 13 17 AGAAGAA TTCTTCTGCTCGGEMX1_2D_18 GAGTCCGAGCAGA ATGTGATGGGAGCCCTT 13 18 AGAAGAA GTTCTTCTGCTCGGEMX1_2D_19 GAGTCCGAGCAGA GATGTGATGGGAGCCCT 13 19 AGAAGAA TGTTCTTCTGCTCGGEMX1_2D_20 GAGTCCGAGCAGA TGATGTGATGGGAGCCC 13 20 AGAAGAATTGTTCTTCTGCTCGG FANCF_2D_10 GGAATCCCTTCTGC CGATCAAGGTGCTGCAG 13 10AGCACC AAGGGA FANCF_2D_11 GGAATCCCTTCTGC GCGATCAAGGTGCTGCA 13 11 AGCACCGAAGGGA FANCF_2D_12 GGAATCCCTTCTGC AGCGATCAAGGTGCTGC 13 12 AGCACCAGAAGGGA FANCF_2D_13 GGAATCCCTTCTGC AAGCGATCAAGGTGCTG 13 13 AGCACCCAGAAGGGA FANCF_2D_14 GGAATCCCTTCTGC AAAGCGATCAAGGTGCT 13 14 AGCACCGCAGAAGGGA FANCF_2D_15 GGAATCCCTTCTGC AAAAGCGATCAAGGTGC 13 15 AGCACCTGCAGAAGGGA FANCF_2D_16 GGAATCCCTTCTGC GAAAAGCGATCAAGGTG 13 16 AGCACCCTGCAGAAGGGA FANCF_2D_17 GGAATCCCTTCTGC GGAAAAGCGATCAAGGT 13 17 AGCACCGCTGCAGAAGGGA FANCF_2D_18 GGAATCCCTTCTGC CGGAAAAGCGATCAAGG 13 18 AGCACCTGCTGCAGAAGGGA FANCF_2D_19 GGAATCCCTTCTGC TCGGAAAAGCGATCAAG 13 19 AGCACCGTGCTGCAGAAGGGA FANCF_2D_20 GGAATCCCTTCTGC CTCGGAAAAGCGATCAA 13 20AGCACC GGTGCTGCAGAAGGGA RNF2_2D_10 GTCATCTTAGTCAT AACACCTCATGTAATGA 1510 TACCTG CTAAGATG RNF2_2D_11 GTCATCTTAGTCAT GAACACCTCATGTAATG 15 11TACCTG ACTAAGATG RNF2_2D_12 GTCATCTTAGTCAT CGAACACCTCATGTAAT 15 12TACCTG GACTAAGATG RNF2_2D_13 GTCATCTTAGTCAT ACGAACACCTCATGTAA 15 13TACCTG TGACTAAGATG RNF2_2D_14 GTCATCTTAGTCAT AACGAACACCTCATGTA 15 14TACCTG ATGACTAAGATG RNF2_2D_15 GTCATCTTAGTCAT CAACGAACACCTCATGT 15 15TACCTG AATGACTAAGATG RNF2_2D_16 GTCATCTTAGTCAT ACAACGAACACCTCATG 15 16TACCTG TAATGACTAAGATG RNF2_2D_17 GTCATCTTAGTCAT TACAACGAACACCTCAT 15 17TACCTG GTAATGACTAAGATG RNF2_2D_18 GTCATCTTAGTCAT TTACAACGAACACCTCA 15 18TACCTG TGTAATGACTAAGATG RNF2_2D_19 GTCATCTTAGTCAT GTTACAACGAACACCTC 1519 TACCTG ATGTAATGACTAAGATG RNF2_2D_20 GTCATCTTAGTCAT AGTTACAACGAACACCT15 20 TACCTG CATGTAATGACTAAGATG HEK4_2D_7 GGCACTGCGGCTGACCCCAACCTCCAGCCG 11 7 GAGGTGG C HEK4_2D_8 GGCACTGCGGCTGAACCCCAACCTCCAGCC 11 8 GAGGTGG GC HEK4_2D_9 GGCACTGCGGCTGTAACCCCAACCTCCAGC 11 9 GAGGTGG CGC HEK4_2D_10 GGCACTGCGGCTGTTAACCCCAACCTCCAG 11 10 GAGGTGG CCGC HEK4_2D_11 GGCACTGCGGCTGTTTAACCCCAACCTCCA 11 11 GAGGTGG GCCGC HEK4_2D_12 GGCACTGCGGCTGCTTTAACCCCAACCTCC 11 12 GAGGTGG AGCCGC HEK4_2D_13 GGCACTGCGGCTGGCTTTAACCCCAACCTC 11 13 GAGGTGG CAGCCGC HEK4_2D_14 GGCACTGCGGCTGCGCTTTAACCCCAACCT 11 14 GAGGTGG CCAGCCGC HEK4_2D_15 GGCACTGCGGCTGCCGCTTTAACCCCAACC 11 15 GAGGTGG TCCAGCCGC HEK4_2D_16 GGCACTGCGGCTGTCCGCTTTAACCCCAAC 11 16 GAGGTGG CTCCAGCCGC HEK4_2D_17 GGCACTGCGGCTGCTCCGCTTTAACCCCAA 11 17 GAGGTGG CCTCCAGCCGC HEK4_2D_18 GGCACTGCGGCTGCTCCGCTTTAACCCCAA 11 18 GAGGTGG CCTCCAGCCGC HEK4_2D_19 GGCACTGCGGCTGCTCCGCTTTAACCCCAA 11 19 GAGGTGG CCTCCAGCCGC

TABLE 3B FIGS. 40A-40C PEgRNA RT SPACER SEQUENCE PBS TEMPLATE(SEQ ID NO: 3′ EXTENSION (SEQ ID LENGTH LENGTH PEGRNA 3104-3112)NOS: 3113-3121) (NT) (NT) RNF2_3B GTCATCTTAGTCAT AACGAACACCTCATGTA 15 14TACCTG ATGACTAAGATG EMX1_3B GAGTCCGAGCAGA ATGGGAGCACTTCTTCT 15 13AGAAGAA TCTGCTCGGAC FANCF_3B GGAATCCCTTCTG GGAAAAGCGATCAAGGT 15 17CAGCACC GCTGCAGAAGGGATT HE3_3B GGCCCAGACTGAG TCTGCCATGACGTGCTC 13 10CACGTGA AGTCTG HEK4_3B GGCACTGCGGCTG TTAACCCCAACCTCCAG 9 10 GAGGTGG CCRNF2_3C_4ATOC GTCATCTTAGTCAT AACGAACACCGCAGGTA 15 14 TACCTG ATGACTAAGATGRNF2_3C_4ATOG GTCATCTTAGTCAT AACGAACACCCCAGGTA 15 14 TACCTG ATGACTAAGATGFANCF_3C_5GTOT GGAATCCCTTCTG GGAAAAGCGATCAAGGT 13 17 CAGCACCGCTGCAGAAGGGA FANCF_3C_7ATOC GGAATCCCTTCTG GGAAAAGCGAGCCAGG 14 17CAGCACC TGCTGCAGAAGGGAT

TABLE 3C FIGS. 40A-40C nicking sgRNA sequences SEQ ID NICKING SGRNASPACER SEQUENCE NO: RNF2_2B_+41 GTCAACCATTAAGCAAAACAT 3122 RNF2_2B_+67GTCTCAGGCTGTGCAGACAAA 3123 EMX1_2B_−116 GGGGCACAGATGAGAAACTC 3124EMX1_2B_−57 GCCGTTTGTACTTTGTCCTC 3125 EMX1_2B_+14 GCGCCACCGGTTGATGTGAT3126 EMX1_2B_+27 GCTTCGTGGCAATGCGCCAC 3127 EMX1_2B_+53GACATCGATGTCCTCCCCAT 3128 EMX1_2B_+80 GTGGTTGCCCACCCTAGTCAT 3129FANCF_2B_−78 GCGACTCTCTGCGTACTGAT 3130 FANCF_2B_−50GCCCTACTTCCGCTTTCACCT 3131 FANCF_2B_−27 GGATTCCATGAGGTGCGCGA 3132FANCF_2B_−17 GCTGCAGAAGGGATTCCATG 3133 FANCF_2B_+21 GCTTGAGACCGCCAGAAGCT3134 FANCF_2B_+48 GGGGTCCCAGGTGCTGACGT 3135 HEK3_2B_−108GCAGAAATAGACTAATTGCA 3136 HEK3_2B_−38 GGATTGACCCAGGCCAGGGC 3137HEK3_2B_+26 GACGCCCTCTGGAGGAAGCA 3138 HEK3_2B_+37 GCTGTCCTGCGACGCCCTC3139 HEK3_2B_+63 GCACATACTAGCCCCTGTCT 3140 HEK3_2B_+90GTCAACCAGTATCCCGGTGC 3141 HEK4_2B_−95 TCCCTTCCTTCCACCCAGCC 3142HEK4_2B_−51 CCCTGCCTGTCATCCTGCTT 3143 HEK4_2B_−26 GCAGTGCCACCGGGGCGCCG3144 HEK4_2B_+52 GCGGGGGCTCAGAGAGGGCA 3145 HEK4_2B_+74GAGACACACACACAGGCCTGG 3146 RNF2_2C_+41 GTCAACCATTAAGCAAAACAT 3147RNF2_2C_4ATOC_+5 GTGAGTTACAACGAACACCGC 3148 RNF2_2C_4ATOG_+5GTGAGTTACAACGAACACCCC 3149 FANCF_2C_+48 GGGGTCCCAGGTGCTGACGT 3150FANCF_2C_5GTOT_+7 GAAGCTCGGAAAAGCGATCA 3151 FANCF_2C_7ATOC_+7GAAGCTCGGAAAAGCGAGCC 3152 HEK3_2C_+90 GTCAACCAGTATCCCGGTGC 3153

TABLE 3D FIGS. 41A-41K PEgRNA RT SPACER SEQUENCE PBS TEMPLATE(SEQ ID NO: 3′ EXTENSION (SEQ ID LENGTH LENGTH PEGRNA 3154-3304)NO: 3305-3455) (NT) (NT) HEK3_4A_1TTOA GGCCCAGACTGAG TCTGCCATCTCGTGCTCA13 10 CACGTGA GTCTG HEK3_4A_1TTOC GGCCCAGACTGAG TCTGCCATCGCGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_1TTOG GGCCCAGACTGAG TCTGCCATCCCGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_2GTOA GGCCCAGACTGAG TCTGCCATTACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_2GTOC GGCCCAGACTGAG TCTGCCATGACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_2GTOT GGCCCAGACTGAG TCTGCCATAACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_3ATOC GGCCCAGACTGAG TCTGCCAGCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_3ATOG GGCCCAGACTGAG TCTGCCACCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_3ATOT GGCCCAGACTGAG TCTGCCAACACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_4TTOA GGCCCAGACTGAG TCTGCCTTCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_4TTOC GGCCCAGACTGAG TCTGCCGTCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_4TTOG GGCCCAGACTGAG TCTGCCCTCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_5GTOA GGCCCAGACTGAG TCTGCTATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_5GTOC GGCCCAGACTGAG TCTGCGATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_5GTOT GGCCCAGACTGAG TCTGCAATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_6GTOA GGCCCAGACTGAG TCTGTCATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_6GTOC GGCCCAGACTGAG TCTGGCATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_6GTOT GGCCCAGACTGAG TCTGACATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_7CTOA GGCCCAGACTGAG TCTTCCATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_7CTOG GGCCCAGACTGAG TCTCCCATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_7CTOT GGCCCAGACTGAG TCTACCATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_8ATOC GGCCCAGACTGAG TCGGCCATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_8ATOG GGCCCAGACTGAG TCCGCCATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4A_8ATOT GGCCCAGACTGAG TCAGCCATCACGTGCTCA 13 10CACGTGA GTCTG HEK3_4B_1TTOA GGCCCAGACTGAG TGGAGGAAGCAGGGCTT 13 34CACGTGA CCTTTCCTCTGCCATCTC GTGCTCAGTCTG HEK3_4B_12GTOC GGCCCAGACTGAGTGGAGGAAGCAGGGCTT 13 34 CACGTGA CCTTTGCTCTGCCATCAC GTGCTCAGTCTGHEK3_4B_14ATOT GGCCCAGACTGAG TGGAGGAAGCAGGGCTT 13 34 CACGTGACCTATCCTCTGCCATCAC GTGCTCAGTCTG HEK3_4B_17GTOC GGCCCAGACTGAGTGGAGGAAGCAGGGCTT 13 34 CACGTGA GCTTTCCTCTGCCATCAC GTGCTCAGTCTGHEK3_4B_20GTOC GGCCCAGACTGAG TGGAGGAAGCAGGGGTT 13 34 CACGTGACCTTTCCTCTGCCATCAC GTGCTCAGTCTG HEK3_4B_23CTOG GGCCCAGACTGAGTGGAGGAAGCACGGCTT 13 34 CACGTGA CCTTTCCTCTGCCATCAC GTGCTCAGTCTGHEK3_4B_24TTOA GGCCCAGACTGAG TGGAGGAAGCTGGGCTT 13 34 CACGTGACCTTTCCTCTGCCATCAC GTGCTCAGTCTG HEK3_4B_26CTOG GGCCCAGACTGAGTGGAGGAACCAGGGCTT 13 34 CACGTGA CCTTTCCTCTGCCATCAC GTGCTCAGTCTGHEK3_4B_30CTOG GGCCCAGACTGAG TGGACGAAGCAGGGCTT 13 34 CACGTGACCTTTCCTCTGCCATCAC GTGCTCAGTCTG HEK3_4B_33CTOG GGCCCAGACTGAGTCGAGGAAGCAGGGCTT 13 34 CACGTGA CCTTTCCTCTGCCATCAC GTGCTCAGTCTGRNF2_4C_1CTOA GTCATCTTAGTCATT AACGAACACCTCATGTAA 15 14 ACCTG TGACTAAGATGRNF2_4C_1CTOG GTCATCTTAGTCATT AACGAACACCTCACGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_1CTOT GTCATCTTAGTCATT AACGAACACCTCAAGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_2TTOA GTCATCTTAGTCATT AACGAACACCTCTGGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_2TTOG GTCATCTTAGTCATT AACGAACACCTCCGGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_3GTOC GTCATCTTAGTCATT AACGAACACCTGAGGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_4ATOC GTCATCTTAGTCATT AACGAACACCGCAGGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_4ATOT GTCATCTTAGTCATT AACGAACACCACAGGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_4ATOG GTCATCTTAGTCATT AACGAACACCCCAGGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_5GTOT GTCATCTTAGTCATT AACGAACACATCAGGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_6GTOA GTCATCTTAGTCATT AACGAACATCTCAGGTA 15 14 ACCTG ATGACTAAGATGRNF2_4C_7TTOC GTCATCTTAGTCATT AACGAACGCCTCAGGTA 15 14 ACCTG ATGACTAAGATGFANCF_4D_1ATOG GGAATCCCTTCTGC GGAAAAGCGATCCAGGC 14 17 AGCACCGCTGCAGAAGGGAT FANCF_4D_1ATOT GGAATCCCTTCTGC GGAAAAGCGATCCAGGA 14 17AGCACC GCTGCAGAAGGGAT FANCF_4D_2CTOA GGAATCCCTTCTGC GGAAAAGCGATCCAGTT 1417 AGCACC GCTGCAGAAGGGAT FANCF_4D_3CTOG GGAATCCCTTCTGC GGAAAAGCGATCCACGT14 17 AGCACC GCTGCAGAAGGGAT FANCF_4D_3CTOT GGAATCCCTTCTGCGGAAAAGCGATCCAAGT 14 17 AGCACC GCTGCAGAAGGGAT FANCF_4D_4TTOAGGAATCCCTTCTGC GGAAAAGCGATCCTGGT 14 17 AGCACC GCTGCAGAAGGGATFANCF_4D_4TTOG GGAATCCCTTCTGC GGAAAAGCGATCCCGGT 14 17 AGCACCGCTGCAGAAGGGAT FANCF_4D_5GTOA GGAATCCCTTCTGC GGAAAAGCGATCTAGGT 14 17AGCACC GCTGCAGAAGGGAT FANCF_4D_6GTOC GGAATCCCTTCTGC GGAAAAGCGATGCAGGT 1417 AGCACC GCTGCAGAAGGGAT FANCF_4D_7ATOC GGAATCCCTTCTGC GGAAAAGCGAGCCAGGT14 17 AGCACC GCTGCAGAAGGGAT FANCF_4D_8TTOC GGAATCCCTTCTGCGGAAAAGCGGTCCAGGT 14 17 AGCACC GCTGCAGAAGGGAT FANCF_4D_10GTOTGGAATCCCTTCTGC GGAAAAGAGATCCAGGT 14 17 AGCACC GCTGCAGAAGGGATEMX1_4E_2ATOC GAGTCCGAGCAGA GTGATGGGAGCCCTGCTT 14 16 AGAAGAACTTCTGCTCGGA EMX1_4E_2ATOT GAGTCCGAGCAGA GTGATGGGAGCCCTACTT 14 16AGAAGAA CTTCTGCTCGGA EMX1_4E_3ATOG GAGTCCGAGCAGA GTGATGGGAGCCCCTCTT 1416 AGAAGAA CTTCTGCTCGGA EMX1_4E_4GTOC GAGTCCGAGCAGA GTGATGGGAGCCGTTCTT14 16 AGAAGAA CTTCTGCTCGGA EMX1_4E_5GTOA GAGTCCGAGCAGAGTGATGGGAGCTCTTCTT 14 16 AGAAGAA CTTCTGCTCGGA EMX1_4E_5GTOTGAGTCCGAGCAGA GTGATGGGAGCACTTCTT 14 16 AGAAGAA CTTCTGCTCGGAEMX1_4E_7CTOA GAGTCCGAGCAGA GTGATGGGATCCCTTCTT 14 16 AGAAGAACTTCTGCTCGGA EMX1_4E_8TTOA GAGTCCGAGCAGA GTGATGGGTGCCCTTCTT 14 16AGAAGAA CTTCTGCTCGGA EMX1_4E_8TTOC GAGTCCGAGCAGA GTGATGGGGGCCCTTCTT 1416 AGAAGAA CTTCTGCTCGGA EMX1_4E_8TTOG GAGTCCGAGCAGA GTGATGGGCGCCCTTCTT14 16 AGAAGAA CTTCTGCTCGGA EMX1_4E_9CTOG GAGTCCGAGCAGAGTGATGGCAGCCCTTCTT 14 16 AGAAGAA CTTCTGCTCGGA EMX1_4E_9CTOTGAGTCCGAGCAGA GTGATGGAAGCCCTTCTT 14 16 AGAAGAA CTTCTGCTCGGARUNX1_4F_1CTOA GCATTTTCAGGAGG TGTCTGAAGCCATCTCTT 15 15 AAGCGACCTCCTGAAAAT RUNX1_4F_1CTOG GCATTTTCAGGAGG TGTCTGAAGCCATCCCTT 15 15AAGCGA CCTCCTGAAAAT RUNX1_4F_1CTOT GCATTTTCAGGAGG TGTCTGAAGCCATCACTT 1515 AAGCGA CCTCCTGAAAAT RUNX1_4F_2GTOA GCATTTTCAGGAGG TGTCTGAAGCCATTGCTT15 15 AAGCGA CCTCCTGAAAAT RUNX1_4F_3ATOC GCATTTTCAGGAGGTGTCTGAAGCCAGCGCTT 15 15 AAGCGA CCTCCTGAAAAT RUNX1_4F_3ATOGGCATTTTCAGGAGG TGTCTGAAGCCACCGCTT 15 15 AAGCGA CCTCCTGAAAATRUNX1_4F_3ATOT GCATTTTCAGGAGG TGTCTGAAGCCAACGCTT 15 15 AAGCGACCTCCTGAAAAT RUNX1_4F_4TTOA GCATTTTCAGGAGG TGTCTGAAGCCTTCGCTT 15 15AAGCGA CCTCCTGAAAAT RUNX1_4F_4TTOC GCATTTTCAGGAGG TGTCTGAAGCCGTCGCTT 1515 AAGCGA CCTCCTGAAAAT RUNX1_4F_4TTOG GCATTTTCAGGAGG TGTCTGAAGCCCTCGCTT15 15 AAGCGA CCTCCTGAAAAT RUNX1_4F_5GTOT GCATTTTCAGGAGGTGTCTGAAGCAATCGCTT 15 15 AAGCGA CCTCCTGAAAAT RUNX1_4F_6GTOCGCATTTTCAGGAGG TGTCTGAAGGCATCGCTT 15 15 AAGCGA CCTCCTGAAAATVEGFA_4G_1TTOA GATGTCTGCAGGCC AATGTGCCATCTGGAGCC 13 22 AGATGACTCTTCTGGCCTGCAGA VEGFA_4G_1TTOC GATGTCTGCAGGCC AATGTGCCATCTGGAGCC 13 22AGATGA CTCGTCTGGCCTGCAGA VEGFA_4G_1TTOG GATGTCTGCAGGCCAATGTGCCATCTGGAGCC 13 22 AGATGA CTCCTCTGGCCTGCAGA VEGFA_4G_2GTOAGATGTCTGCAGGCC AATGTGCCATCTGGAGCC 13 22 AGATGA CTTATCTGGCCTGCAGAVEGFA_4G_3ATOC GATGTCTGCAGGCC AATGTGCCATCTGGAGCC 13 22 AGATGACGCATCTGGCCTGCAGA VEGFA_4G_3ATOG GATGTCTGCAGGCC AATGTGCCATCTGGAGCC 13 22AGATGA CCCATCTGGCCTGCAGA VEGFA_4G_3ATOT GATGTCTGCAGGCCAATGTGCCATCTGGAGCC 13 22 AGATGA CACATCTGGCCTGCAGA VEGFA_4G_5GTOTGATGTCTGCAGGCC AATGTGCCATCTGGAGCA 13 22 AGATGA CTCATCTGGCCTGCAGAVEGFA_4G_6GTOC GATGTCTGCAGGCC AATGTGCCATCTGGAGGC 13 22 AGATGACTCATCTGGCCTGCAGA VEGFA_4G_7CTOA GATGTCTGCAGGCC AATGTGCCATCTGGATCC 13 22AGATGA CTCATCTGGCCTGCAGA VEGFA_4G_7CTOT GATGTCTGCAGGCCAATGTGCCATCTGGAACC 13 22 AGATGA CTCATCTGGCCTGCAGA VEGFA_4G_9CTOGGATGTCTGCAGGCC AATGTGCCATCTGCAGCC 13 22 AGATGA CTCATCTGGCCTGCAGADNMT1_4H_1ATOC GATTCCTGGTGCCA GTCACCCCTGGTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_1ATOG GATTCCTGGTGCCA GTCACCCCTGCTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_2CTOA GATTCCTGGTGCCA GTCACCCCTTTTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_2CTOG GATTCCTGGTGCCA GTCACCCCTCTTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_2CTOT GATTCCTGGTGCCA GTCACCCCTATTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_3ATOT GATTCCTGGTGCCA GTCACCCCAGTTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_4GTOA GATTCCTGGTGCCA GTCACCCTTGTTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_5GTOT GATTCCTGGTGCCA GTCACCACTGTTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_6GTOC GATTCCTGGTGCCA GTCACGCCTGTTTCTGGC 13 11 GAAACA ACCAGGDNMT1_4H_8TTOA GATTCCTGGTGCCA GCCCTCCCGTCTCCCCTG 13 19 GAAACATTTCTGGCACCAGG DNMT1_4H_8TTOC GATTCCTGGTGCCA GCCCTCCCGTCGCCCCTG 13 19GAAACA TTTCTGGCACCAGG DNMT1_4H_8TTOG GATTCCTGGTGCCA GCCCTCCCGTCCCCCCTG13 19 GAAACA TTTCTGGCACCAGG HEK3_4J_DEL1-5 GGCCCAGACTGAGTGGAGGAAGCAGGGCTT 13 29 CACGTGA CCTTTCCTCTGCCGTGCT CAGTCTGHEK3_4J_DEL1-10 GGCCCAGACTGAG TGGAGGAAGCAGGGCTT 13 24 CACGTGACCTTTCCCGTGCTCAGTC TG HEK3_4J_DEL1-15 GGCCCAGACTGAG TGGAGGAAGCAGGGCTT 1319 CACGTGA CCCGTGCTCAGTCTG HEK3_4J_DEL1-25 GGCCCAGACTGAGTGTCCTGCGACGCCCTCT 13 26 CACGTGA GGAGGAAGCGTGCTCAG TCTG HEK3_4J_DEL1-30GGCCCAGACTGAG TGTCCTGCGACGCCCTCT 13 21 CACGTGA GGACGTGCTCAGTCTGHEK3_4J_DEL1-80 GGCCCAGACTGAG AGTATCCCGGTGCAGGA 13 20 CACGTGAGCTCGTGCTCAGTCTG HEK3_4I_1AINS GGCCCAGACTGAG TCTGCCATCATCGTGCTC 13 11CACGTGA AGTCTG HEK3_4I_1CTTINS GGCCCAGACTGAG TCTGCCATCAAAGCGTGC 13 13CACGTGA TCAGTCTG HEK3_4I_1TDEL GGCCCAGACTGAG TCTGCCATCCGTGCTCAG 13 9CACGTGA TCTG HEK3_4I_1- GGCCCAGACTGAG TGGAGGAAGCAGGGCTT 13 31 3TGADELCACGTGA CCTTTCCTCTGCCACGTG CTCAGTCTG RNF2_4I_1TINS GTCATCTTAGTCATTAACGAACACCTCAGAGT 15 15 ACCTG AATGACTAAGATG RNF2_4I_1GTAINSGTCATCTTAGTCATT AACGAACACCTCAGTAC 15 17 ACCTG GTAATGACTAAGATGRNF2_4I_4ADEL GTCATCTTAGTCATT AACGAACACCCAGGTAA 15 13 ACCTG TGACTAAGATGRNF2_4I_3-5GAGDEL GTCATCTTAGTCATT AACGAACACAGGTAATG 15 11 ACCTGACTAAGATG FANCF_4I_3CINS GGAATCCCTTCTGC GGAAAAGCGATCCAGGG 14 18 AGCACCTGCTGCAGAAGGGAT FANCF_4I_4GATINS GGAATCCCTTCTGC GGAAAAGCGATCCAATC 14 20AGCACC GGTGCTGCAGAAGGGAT FANCF_4I_6GDEL GGAATCCCTTCTGC GGAAAAGCGATCAGGTG14 16 AGCACC CTGCAGAAGGGAT FANCF_4I_5- GGAATCCCTTCTGC GGAAAAGCGAAGGTGCT14 14 7GGADEL AGCACC GCAGAAGGGAT EMX1_4I_6TINS GAGTCCGAGCAGAGTGATGGGAGCACCTTCT 14 17 AGAAGAA TCTTCTGCTCGGA EMX1_4I_1TGCINSGAGTCCGAGCAGA GTGATGGGAGCCCTTCGC 14 19 AGAAGAA ATTCTTCTGCTCGGAEMX1_4I_5GDEL GAGTCCGAGCAGA GTGATGGGAGCCTTCTTC 14 15 AGAAGAA TTCTGCTCGGAEMX1_4I_4- GAGTCCGAGCAGA GTGATGGGAGTTCTTCTT 14 13 6GGGDEL AGAAGAACTGCTCGGA RUNX1_4I_1CINS GCATTTTCAGGAGG TGTCTGAAGCCATCGGCT 15 16 AAGCGATCCTCCTGAAAAT RUNX1_4I_1ATGINS GCATTTTCAGGAGG TGTCTGAAGCCATCCATG 15 18AAGCGA CTTCCTCCTGAAAAT RUNX1_4I_2GDEL GCATTTTCAGGAGG TGTCTGAAGCCATGCTTC15 14 AAGCGA CTCCTGAAAAT RUNX1_4I_2- GCATTTTCAGGAGG TGTCTGAAGCCGCTTCCT15 12 4GATDEL AAGCGA CCTGAAAAT VEGFA_4I_4CINS GATGTCTGCAGGCCAATGTGCCATCTGGAGCC 13 23 AGATGA GCTCATCTGGCCTGCAGA VEGFA_4I_2ACAINSGATGTCTGCAGGCC AATGTGCCATCTGGAGCC 13 25 AGATGA CTTGTCATCTGGCCTGCA GAVEGFA_4I_3ADEL GATGTCTGCAGGCC AATGTGCCATCTGGAGCC 13 21 AGATGACCATCTGGCCTGCAGA VEGFA_4I_2- GATGTCTGCAGGCC AATGTGCCATCTGGAGCC 13 194GAGDEL AGATGA ATCTGGCCTGCAGA DNMT1_4I_4CINS GATTCCTGGTGCCATCCCGTCACCCGCTGTTT 13 16 GAAACA CTGGCACCAGG DNMT1_4I_1TCAINSGATTCCTGGTGCCA TCCCGTCACCCCTGTGAT 13 18 GAAACA TTCTGGCACCAGGDNMT1_4I_3ADEL GATTCCTGGTGCCA TCCCGTCACCCCGTTTCT 13 14 GAAACA GGCACCAGGDNMT1_4I_3- GATTCCTGGTGCCA TCCCGTCACCGTTTCTGG 13 12 5AGGDEL GAAACACACCAGG HEK3_4K_1CTTINS_5GDEL GGCCCAGACTGAG TGGAGGAAGCAGGGCTT 13 36CACGTGA CCTTTCCTCTGCATCAAA GCGTGCTCAGTCTG HEK3_4K_1CTTINS_2GTOCGGCCCAGACTGAG TGGAGGAAGCAGGGCTT 13 37 CACGTGA CCTTTCCTCTGCCATGAAAGCGTGCTCAGTCTG HEK3_4K_3TDEL_5GTOC GGCCCAGACTGAG TGGAGGAAGCAGGGCTT 1333 CACGTGA CCTTTCCTCTGCGATCCG TGCTCAGTCTG HEK3_4K_3GTOC_6GTOTGGCCCAGACTGAG TGGAGGAAGCAGGGCTT 13 34 CACGTGA CCTTTCCTCTGACATGACGTGCTCAGTCTG RNF2_4K_2AAINS_3- GTCATCTTAGTCATT AACGAACACCATTGGTA 15 144GADEL ACCTG ATGACTAAGATG RNF2_4K_1AINS_5GTOC GTCATCTTAGTCATTAACGAACACGTCAGTGT 15 15 ACCTG AATGACTAAGATG RNF2_4K_1- GTCATCTTAGTCATTAACGAACAACTCGTAAT 15 12 2CTDEL_6GTOT ACCTG GACTAAGATGRNF2_4K_1CTOA_5GTOT GTCATCTTAGTCATT AACGAACACATCATGTAA 15 14 ACCTGTGACTAAGATG FANCF_4K_1TINS_3- GGAATCCCTTCTGC GGAAAAGCGATCGGTAG 14 164TGDEL AGCACC CTGCAGAAGGGAT FANCF_4K_1TINS_6GTOA GGAATCCCTTCTGCGGAAAAGCGATTCAGGT 14 18 AGCACC AGCTGCAGAAGGGAT FANCF_4K_2CDEL_5GTOTGGAATCCCTTCTGC GGAAAAGCGATCAAGTG 14 16 AGCACC CTGCAGAAGGGAT

TABLE 3E FIGS. 41A-41K nicking sgRNA SPACER SEQUENCE NICKING SGRNA(SEQ ID NOS: 3456-3463) HEK3_4A_+90 GTCAACCAGTATCCCGGTGC HEK3_4B_+90GTCAACCAGTATCCCGGTGC RNF2_4C_+41 GTCAACCATTAAGCAAAACAT FANCF_4D_+48GGGGTCCCAGGTGCTGACGT EMX1_4E_+53 GACATCGATGTCCTCCCCAT RUNX1_4F_+38GATGAAGCACTGTGGGTACGA VEGFA_4G_+57 GATGTACAGAGAGCCCAGGGC DNMT1_4H_+49GCCCTTCAGCTAAAATAAAGG

TABLE 3F FIGS. 42A-42H PEgRNA RT SPACER PBS TEMPLATE SEQUENCE (SEQ3′ EXTENSION (SEQ ID LENGTH LENGTH PEGRNA ID NOS: 3464-3478)NOS: 3479-3493) (NT) (NT) HEK3_5A_C3 GGCCCAGACTGA TCTGTCATCACGTGCTCAGT13 10 GCACGTGA CTG HEK3_5A_C4 GGCCCAGACTGA TCTGCTATCACGTGCTCAGT 13 10GCACGTGA CTG HEK3_5A_C7 GGCCCAGACTGA TCTGCCATTACGTGCTCAGT 13 10 GCACGTGACTG FANCF_5A_C3 GGAATCCCTTCTG GGAAAAGTGATCCAGGTGC 14 17 CAGCACCTGCAGAAGGGAT FANCF_5A_C7 GGAATCCCTTCTG GGAAAAGCGATTCAGGTGC 14 17 CAGCACCTGCAGAAGGGAT FANCF_5A_C8 GGAATCCCTTCTG GGAAAAGCGATCTAGGTGC 14 17 CAGCACCTGCAGAAGGGAT EMX1_5A_C5 GAGTCCGAGCAG GTGATGGGAGTCCTTCTTCT 14 16 AAGAAGAATCTGCTCGGA EMX1_5A_C6 GAGTCCGAGCAG GTGATGGGAGCTCTTCTTCT 14 16 AAGAAGAATCTGCTCGGA EMX1_5A_C7 GAGTCCGAGCAG GTGATGGGAGCCTTTCTTCT 14 16 AAGAAGAATCTGCTCGGA EMX1_5C_C5_6 GAGTCCGAGCAG GTGATGGGAGTTCTTCTTCT 14 16 AAGAAGAATCTGCTCGGA EMX1_5C_C5_7 GAGTCCGAGCAG GTGATGGGAGTCTTTCTTCT 14 16 AAGAAGAATCTGCTCGGA EMX1_5C_C6_7 GAGTCCGAGCAG GTGATGGGAGCTTTTCTTCT 14 16 AAGAAGAATCTGCTCGGA EMX1_5C_C5_6_7 GAGTCCGAGCAG GTGATGGGAGTTTTTCTTCT 14 16AAGAAGAA TCTGCTCGGA HEK3_5D_A5 GGCCCAGACTGA TCTGCCGTCACGTGCTCAGT 13 10GCACGTGA CTG HEK3_5D_A8 GGCCCAGACTGA TCTGCCATCGCGTGCTCAGT 13 10 GCACGTGACTG

TABLE 3G FIGS. 42A-42H nicking sgRNA POSSIBLE SPACER POSSIBLE SPACERNICKING SEQUENCE SEQUENCE (SEQ ID SGRNA (SEQ ID NOS: 616-618)NOS: 619-621) HEK3_5A- GTCAACCAGTATCCCGGTGC GTCAACCAGTATCCCGGTGC F_+90FANCF_5A- GGGGTCCCAGGTGCTGACGT GATGTACAGAGAGCCCAGGG F_+48 C EMX1_5A-GATGTACAGAGAGCCCAGGGC GGGGTCCCAGGTGCTGACGT F_+57

TABLE 3H FIGS. 42A-42H base editing sgRNA BASE EDITING SGRNASPACER SEQUENCE HEK3_5A-F_BE GTGCCATCACGTGCTCAGTCT (SEQ ID NO: 455)FANCF_5A-F_BE GAGCGATCCAGGTGCTGCAGA (SEQ ID NO: 456) EMX1_5A-F_BEGGAGCCCTTCTTCTTCTGCT (SEQ ID NO: 457)

TABLE 3I FIGS. 42A-42H on-target sgRNA ON-TARGET SGRNA SPACER SEQUENCEHEK3_5G GGCCCAGACTGAGCACGTGA (SEQ ID NO: 510) HEK4_5GGGCACTGCGGCTGGAGGTGG (SEQ ID NO: 511) EMX1_5G GAGTCCGAGCAGAAGAAGAA(SEQ ID NO: 512) FANCF_5G GGAATCCCTTCTGCAGCACC (SEQ ID NO: 513)

TABLE 3J FIGS. 42A-42H on-target PEgRNA RT PBS TEMPLATE ON-TARGETSPACER SEQUENCE 3′ EXTENSION (SEQ ID LENGTH LENGTH PEGRNA(SEQ ID NO: 663-677) NO: 678-692) (NT) (NT) HEK3_5G- GGCCCAGACTGAGCTCTGCCATCTCGTGCTCA 13 10 H_PEGRNA_1 ACGTGA GTCTG HEK3_5G- GGCCCAGACTGAGCTCTGCCATCAAAGCGTG 13 13 H_PEGRNA_2 ACGTGA CTCAGTCTG HEK3_5G-GGCCCAGACTGAGC TCTGCCATCCGTGCTCA 13 9 H_PEGRNA_3 ACGTGA GTCTG HEK3_5G-GGCCCAGACTGAGC TCTGCGATCACGTGCTC 13 10 H_PEGRNA_4 ACGTGA AGTCTG HEK4_5G-GGCACTGCGGCTGG TTAACGCCCACCTCCAG 9 10 H_PEGRNA_1 AGGTGG CC HEK4_5G-GGCACTGCGGCTGG TTAACCCCCCCCTCCAG 9 10 H_PEGRNA_2 AGGTGG CC HEK4_5G-GGCACTGCGGCTGG TTAACCCCTTACACCTCC 9 13 H_PEGRNA_3 AGGTGG AGCC HEK4_5G-GGCACTGCGGCTGG TTAACCCCCCCTCCAGC 9 9 H_PEGRNA_4 AGGTGG C EMX1_5G-GAGTCCGAGCAGAA GTGATGGGAGCACTTCT 14 16 H_PEGRNA_1 GAAGAA TCTTCTGCTCGGAEMX1_5G- GAGTCCGAGCAGAA GTGATGGGAGCCCTGCT 14 16 H_PEGRNA_2 GAAGAATCTTCTGCTCGGA EMX1_5G- GAGTCCGAGCAGAA GTGATGGGAGCCCTTCG 14 19 H_PEGRNA_3GAAGAA CATTCTTCTGCTCGGA EMX1_5G- GAGTCCGAGCAGAA GTGATGGGAGTTCTTCTT 14 13H_PEGRNA_4 GAAGAA CTGCTCGGA FANCF_5G- GGAATCCCTTCTGCA GGAAAAGCGATGCAGGT14 17 H_PEGRNA_1 GCACC GCTGCAGAAGGGAT FANCF_5G- GGAATCCCTTCTGCAGGAAAAGCGATCCAGGC 14 17 H_PEGRNA_2 GCACC GCTGCAGAAGGGAT FANCF_5G-GGAATCCCTTCTGCA GGAAAAGCGATCCAATC 14 20 H_PEGRNA_3 GCACCGGTGCTGCAGAAGGGAT

TABLE 3K FIGs. 49A-49BPEgRNA RT SPACER PBS TEMPLATE SEQUENCE (SEQ3′ EXTENSION (SEQ ID LENGTH LENGTH PEGRNA ID NO: 3494-3521)NO: 3522-3540) (NT) (NT) HEK3_6A_2GTOC GGCCCAGACTG TCTGCCATGACGTGCTC 1310 AGCACGTGA AGTCTG HEK3_6A_2GTOC GGCCCAGACTG AGCACGTGA EMX1_6A_3GTOCGAGTCCGAGCA ATGGGAGCCCTTGTTCT 13 13 GAAGAAGAA TCTGCTCGG EMX1_6A_3GTOCGAGTCCGAGCA GAAGAAGAA FANCF_6A_5GTOT GGAATCCCTTCT AAAAGCGATCAAGGTGC 1315 GCAGCACC TGCAGAAGGGA FANCF_6A_5GTOT GGAATCCCTTCT GCAGCACCHEK3_6A_1HIS6INS GGCCCAGACTG TGGAGGAAGCAGGGCTT 13 52 AGCACGTGACCTTTCCTCTGCCATCAA TGATGGTGATGATGGTG CGTGCTCAGTCTG HEK3_6A_1HIS6INSGGCCCAGACTG AGCACGTGA HEK3_6A_5GTOT GGCCCAGACTG TCTGCAATCACGTGCTC 13 10AGCACGTGA AGTCTG HEK3_6A_5GTOT GGCCCAGACTG AGCACGTGA HEK3_6A_1CTTINSGGCCCAGACTG TCTGCCATCAAAGCGTG 13 10 AGCACGTGA CTCAGTCTG HEK3_6A_1CTTINSGGCCCAGACTG AGCACGTGA HBB_6B_INSALL GCATGGTGCAC AGACTTCTCCACAGGAG 13 14CTGACTCCTG TCAGGTGCAC HBB_6B_INSALL GCATGGTGCAC CTGACTCCTGHBB_6B_CORRECT GCATGGTGCAC AGACTTCTCCTCAGGAG 13 14 CTGACTCCTG TCAGGTGCACHBB_6B_CORRECT GCATGGTGCAC CTGACTCCTG HBB_6B_CORRECT_W_SILENTGCATGGTGCAC AGACTTCTCTTCAGGAG 13 14 CTGACTCCTG TCAGGTGCACHBB_6B_CORRECT_W_SILENT GCATGGTGCAC CTGACTCCTG HEXA_6B_INSTALLGTACCTGAACC AGTCAGGGCCATAGGAT 12 14 GTATATCCTA AGATATACGGTTCHEXA_6B_CORRECT GATCCTTCCAGT ACCTGAACCGTATATCCT 10 21 CAGGGCCATATGGCCCTGACTG HEXA_6B_CORRECT_W_SILENT GATCCTTCCAGT GTACCTGAACCGTATATC 927 CAGGGCCAT TTATGGCCCTGACT PRNP_6C GCAGTGGTGGG ATGTAGACGCCAAGGCC 12 12GGGCCTTGG CCCCACC HEK3_6E- GGCCCAGACTG TCTGCCATCCCGTGCTC 13 10 G_1TTOGAGCACGTGA AGTCTG HEK3_6E- GGCCCAGACTG TCTGCCATCAAAGCGTG 13 10 G_1CTTINSAGCACGTGA CTCAGTCTG RNF2_6E- GTCATCTTAGTC AACGAACACCTCACGTA 15 14G_1CTOG ATTACCTG ATGACTAAGATG HBB_6E- GCATGGTGCAC AGACTTCTCCACAGGAGG_4ATOT CTGACTCCTG TCAGGTGCAC 13 14 HEK3_6H_1HIS6INS GGCCCAGACTGTGGAGGAAGCAGGGCTT 13 52 AGCACGTGA CCTTTCCTCTGCCATCAA TGATGGTGATGATGGTGCGTGCTCAGTCTG HEK3_6H_1FLAGINS GGCCCAGACTG TGGAGGAAGCAGGGCTT 13 58AGCACGTGA CCTTTCCTCTGCCATCAC TTATCGTCGTCATCCTTG TAATCCGTGCTCAGTCT G

TABLE 3L FIGS. 47A-74D PEgRNA RT SPACER SEQUENCE PBS TEMPLATE(SEQ ID NO: 3′ EXTENSION SEQUENCE LENGTH LENGTH PEGRNA 3541-3547)(SEQ ID NO: 3549-3556) (NT) (NT) HEK3_ED4B_1TDEL GGCCCAGACTGAGCTCTGCCATCCGTGCTCAG 13 9 ACGTGA TCTG HEK3_ED4B_1AINS GGCCCAGACTGAGCTCTGCCATCATCGTGCTC 13 11 ACGTGA AGTCTG HEK3_ED4B_1CTTINS GGCCCAGACTGAGCTCTGCCATCAAAGCGTGC 13 13 ACGTGA TCAGTCTG HEK3_ED4C_2GTOC GGCCCAGACTGAGCTCTGCCATGACGTGCTCA 13 10 ACGTGA GTCTG HEK3_ED4D_1FLAGINS GGCCCAGACTGAGCTGGAGGAAGCAGGGCTT 13 58 ACGTGA CCTTTCCTCTGCCATCAC TTATCGTCGTCATCCTTGTAATCCGTGCTCAGTCTG RNF2_ED4E_1CTOA GTCATCTTAGTCATT AACGAACACCTCATGTAA 1514 ACCTG TGACTAAGATG EMX1_ED4F_1GTOC GAGTCCGAGCAGAA ATGGGAGCCCTTGTTCTT13 13 GAAGAA CTGCTCGG HBB_ED4G_2TTOA GTAACGGCAGACTT ATCTGACTCCTGTGGAGA12 14 CTCCTC AGTCTGCC

TABLE 3M FIGS. 48A-48C PEgRNA RT SPACER PBS TEMPLATE SEQUENCE (SEQ3' EXTENSION SEQUENCE LENGTH LENGTH PEGRNA ID NO: 3557-3627)(SEQ ID NO: 3628-3698) (NT) (NT) VEGFA_ED5A_31 GATGTCTGCAGGCCCCTCTGACAATGTGCCATC 13 31 CAGATGA TGGAGCACTCATCTGGCCTG CAGAVEGFA_ED5A_30 GATGTCTGCAGGC CCTCTGACAATGTGCCATCT 13 30 CAGATGAGGAGCACTCATCTGGCCTGC AGA VEGFA_ED5A_29 GATGTCTGCAGGCCTCTGACAATGTGCCATCTG 13 29 CAGATGA GAGCACTCATCTGGCCTGCA GA VEGFA_ED5A_28GATGTCTGCAGGC TCTGACAATGTGCCATCTGG 13 28 CAGATGA AGCACTCATCTGGCCTGCAG AVEGFA_ED5A_27 GATGTCTGCAGGC CTGACAATGTGCCATCTGGA 13 27 CAGATGAGCACTCATCTGGCCTGCAGA VEGFA_ED5A_26 GATGTCTGCAGGC TGACAATGTGCCATCTGGAG 1326 CAGATGA CACTCATCTGGCCTGCAGA VEGFA_ED5A_25 GATGTCTGCAGGCGACAATGTGCCATCTGGAGC 13 25 CAGATGA ACTCATCTGGCCTGCAGA VEGFA_ED5A_24GATGTCTGCAGGC ACAATGTGCCATCTGGAGCA 13 24 CAGATGA CTCATCTGGCCTGCAGAVEGFA_ED5A_23 GATGTCTGCAGGC CAATGTGCCATCTGGAGCAC 13 23 CAGATGATCATCTGGCCTGCAGA VEGFA_ED5A_22 GATGTCTGCAGGC AATGTGCCATCTGGAGCACT 13 22CAGATGA CATCTGGCCTGCAGA VEGFA_ED5A_21 GATGTCTGCAGGC ATGTGCCATCTGGAGCACTC13 21 CAGATGA ATCTGGCCTGCAGA VEGFA_ED5A_20 GATGTCTGCAGGCTGTGCCATCTGGAGCACTCA 13 20 CAGATGA TCTGGCCTGCAGA VEGFA_ED5A_19GATGTCTGCAGGC GTGCCATCTGGAGCACTCAT 13 19 CAGATGA CTGGCCTGCAGAVEGFA_ED5A_18 GATGTCTGCAGGC TGCCATCTGGAGCACTCATC 13 18 CAGATGATGGCCTGCAGA VEGFA_ED5A_17 GATGTCTGCAGGC GCCATCTGGAGCACTCATCT 13 17CAGATGA GGCCTGCAGA VEGFA_ED5A_16 GATGTCTGCAGGC CCATCTGGAGCACTCATCTG 1316 CAGATGA GCCTGCAGA VEGFA_ED5A_15 GATGTCTGCAGGC CATCTGGAGCACTCATCTGG 1315 CAGATGA CCTGCAGA VEGFA_ED5A_14 GATGTCTGCAGGC ATCTGGAGCACTCATCTGGC 1314 CAGATGA CTGCAGA VEGFA_ED5A_13 GATGTCTGCAGGC TCTGGAGCACTCATCTGGCC 1313 CAGATGA TGCAGA VEGFA_ED5A_12 GATGTCTGCAGGC CTGGAGCACTCATCTGGCCT 13 12CAGATGA GCAGA VEGFA_ED5A_11 GATGTCTGCAGGC TGGAGCACTCATCTGGCCTG 13 11CAGATGA CAGA VEGFA_ED5A_10 GATGTCTGCAGGC GGAGCACTCATCTGGCCTGC 13 10CAGATGA AGA VEGFA_ED5A_9 GATGTCTGCAGGC GAGCACTCATCTGGCCTGCA 13 9 CAGATGAGA VEGFA_ED5A_8 GATGTCTGCAGGC AGCACTCATCTGGCCTGCAG 13 8 CAGATGA ADNMT1_ED5B_31 GATTCCTGGTGCC AGGACTAGTTCTGCCCTCCC 13 31 AGAAACAGTCACCACTGTTTCTGGCAC CAGG DNMT1_ED5B_30 GATTCCTGGTGCCGGACTAGTTCTGCCCTCCCG 13 30 AGAAACA TCACCACTGTTTCTGGCACC AGGDNMT1_ED5B_29 GATTCCTGGTGCC GACTAGTTCTGCCCTCCCGT 13 29 AGAAACACACCACTGTTTCTGGCACCA GG DNMT1_ED5B_28 GATTCCTGGTGCC ACTAGTTCTGCCCTCCCGTC13 28 AGAAACA ACCACTGTTTCTGGCACCAG G DNMT1_ED5B_27 GATTCCTGGTGCCCTAGTTCTGCCCTCCCGTCA 13 27 AGAAACA CCACTGTTTCTGGCACCAGG DNMT1_ED5B_26GATTCCTGGTGCC TAGTTCTGCCCTCCCGTCAC 13 26 AGAAACA CACTGTTTCTGGCACCAGGDNMT1_ED5B_25 GATTCCTGGTGCC AGTTCTGCCCTCCCGTCACC 13 25 AGAAACAACTGTTTCTGGCACCAGG DNMT1_ED5B_24 GATTCCTGGTGCC GTTCTGCCCTCCCGTCACCA 1324 AGAAACA CTGTTTCTGGCACCAGG DNMT1_ED5B_23 GATTCCTGGTGCCTTCTGCCCTCCCGTCACCAC 13 23 AGAAACA TGTTTCTGGCACCAGG DNMT1_ED5B_22GATTCCTGGTGCC TCTGCCCTCCCGTCACCACT 13 22 AGAAACA GTTTCTGGCACCAGGDNMT1_ED5B_21 GATTCCTGGTGCC CTGCCCTCCCGTCACCACTG 13 21 AGAAACATTTCTGGCACCAGG DNMT1_ED5B_20 GATTCCTGGTGCC TGCCCTCCCGTCACCACTGT 13 20AGAAACA TTCTGGCACCAGG DNMT1_ED5B_19 GATTCCTGGTGCC GCCCTCCCGTCACCACTGTT13 19 AGAAACA TCTGGCACCAGG DNMT1_ED5B_18 GATTCCTGGTGCCCCCTCCCGTCACCACTGTTT 13 18 AGAAACA CTGGCACCAGG DNMT1_ED5B_17GATTCCTGGTGCC CCTCCCGTCACCACTGTTTC 13 17 AGAAACA TGGCACCAGGDNMT1_ED5B_16 GATTCCTGGTGCC CTCCCGTCACCACTGTTTCT 13 16 AGAAACA GGCACCAGGDNMT1_ED5B_15 GATTCCTGGTGCC TCCCGTCACCACTGTTTCTG 13 15 AGAAACA GCACCAGGDNMT1_ED5B_14 GATTCCTGGTGCC CCCGTCACCACTGTTTCTGG 13 14 AGAAACA CACCAGGDNMT1_ED5B_13 GATTCCTGGTGCC CCGTCACCACTGTTTCTGGC 13 13 AGAAACA ACCAGGDNMT1_ED5B_12 GATTCCTGGTGCC CGTCACCACTGTTTCTGGCA 13 12 AGAAACA CCAGGDNMT1_ED5B_11 GATTCCTGGTGCC GTCACCACTGTTTCTGGCAC 13 11 AGAAACA CAGGDNMT1_ED5B_10 GATTCCTGGTGCC TCACCACTGTTTCTGGCACC 13 10 AGAAACA AGGDNMT1_ED5B_9 GATTCCTGGTGCC CACCACTGTTTCTGGCACCA 13 9 AGAAACA GGDNMT1_ED5B_8 GATTCCTGGTGCC ACCACTGTTTCTGGCACCAG 13 8 AGAAACA GRUNX1_ED5C_31 GCATTTTCAGGAG AATGACTCAAATATGCTGTC 15 31 GAAGCGATGAAGCAATCGCTTCCTCCT GAAAAT RUNX1_ED5C_30 GCATTTTCAGGAGATGACTCAAATATGCTGTCT 15 30 GAAGCGA GAAGCAATCGCTTCCTCCTG AAAATRUNX1_ED5C_29 GCATTTTCAGGAG TGACTCAAATATGCTGTCTG 15 29 GAAGCGAAAGCAATCGCTTCCTCCTGA AAAT RUNX1_ED5C_28 GCATTTTCAGGAGGACTCAAATATGCTGTCTGA 15 28 GAAGCGA AGCAATCGCTTCCTCCTGAA AATRUNX1_ED5C_27 GCATTTTCAGGAG ACTCAAATATGCTGTCTGAA 15 27 GAAGCGAGCAATCGCTTCCTCCTGAAA AT RUNX1_ED5C_26 GCATTTTCAGGAG CTCAAATATGCTGTCTGAAG15 26 GAAGCGA CAATCGCTTCCTCCTGAAAA T RUNX1_ED5C_25 GCATTTTCAGGAGTCAAATATGCTGTCTGAAGC 15 25 GAAGCGA AATCGCTTCCTCCTGAAAAT RUNX1_ED5C_24GCATTTTCAGGAG CAAATATGCTGTCTGAAGCA 15 24 GAAGCGA ATCGCTTCCTCCTGAAAATRUNX1_ED5C_23 GCATTTTCAGGAG AAATATGCTGTCTGAAGCAA 15 23 GAAGCGATCGCTTCCTCCTGAAAAT RUNX1_ED5C_22 GCATTTTCAGGAG AATATGCTGTCTGAAGCAAT 1522 GAAGCGA CGCTTCCTCCTGAAAAT RUNX1_ED5C_21 GCATTTTCAGGAGATATGCTGTCTGAAGCAATC 15 21 GAAGCGA GCTTCCTCCTGAAAAT RUNX1_ED5C_20GCATTTTCAGGAG TATGCTGTCTGAAGCAATCG 15 20 GAAGCGA CTTCCTCCTGAAAATRUNX1_ED5C_19 GCATTTTCAGGAG ATGCTGTCTGAAGCAATCGC 15 19 GAAGCGATTCCTCCTGAAAAT RUNX1_ED5C_18 GCATTTTCAGGAG TGCTGTCTGAAGCAATCGCT 15 18GAAGCGA TCCTCCTGAAAAT RUNX1_ED5C_17 GCATTTTCAGGAG GCTGTCTGAAGCAATCGCTT15 17 GAAGCGA CCTCCTGAAAAT RUNX1_ED5C_16 GCATTTTCAGGAGCTGTCTGAAGCAATCGCTTC 15 16 GAAGCGA CTCCTGAAAAT RUNX1_ED5C_15GCATTTTCAGGAG TGTCTGAAGCAATCGCTTCC 15 15 GAAGCGA TCCTGAAAATRUNX1_ED5C_14 GCATTTTCAGGAG GTCTGAAGCAATCGCTTCCT 15 14 GAAGCGA CCTGAAAATRUNX1_ED5C_13 GCATTTTCAGGAG TCTGAAGCAATCGCTTCCTC 15 13 GAAGCGA CTGAAAATRUNX1_ED5C_12 GCATTTTCAGGAG CTGAAGCAATCGCTTCCTCC 15 12 GAAGCGA TGAAAATRUNX1_ED5C_11 GCATTTTCAGGAG TGAAGCAATCGCTTCCTCCT 15 11 GAAGCGA GAAAATRUNX1_ED5C_10 GCATTTTCAGGAG GAAGCAATCGCTTCCTCCTG 15 10 GAAGCGA AAAATRUNX1_ED5C_9 GCATTTTCAGGAG AAGCAATCGCTTCCTCCTGA 15 9 GAAGCGA AAAT

TABLE 3N FIGs. 48A-48C PEgRNA RT PBS TEMPLATE 3′ EXTENSION LENGTH LENGTHPEGRNA SPACER SEQUENCE SEQUENCE (NT) (NT) HEK3_ED6_5GTOA GGCCCAGACTGAGCTCTGCTATCACGTGCT 13 10 ACGTGA (SEQ ID CAGTCTG (SEQ ID NO: NO: 393) 394)

TABLE 3O FIGs. 48A-48C nicking sgRNA NICKING SGRNA SPACER SEQUENCEHEK3_ED6_+63 GCACATACTAGCCCCTGTCT (SEQ ID NO: 395)

TABLE 3P FIGs. 50A-50B PEgRNA RT PBS TEMPLATE SPACER (SEQ ID3′ EXTENSION (5′ TO 3′) LENGTH LENGTH PEGRNA NO: 3699-3754)(SEQ ID NO: 3755-3810) (NT) (NT) HBB GTAACGGCAGACAGACTTCTCCTCAGGAGTCAGGT 12 14 3.5 TTCTCCAC GCAC HBB GCATGGTGCACCTAGACTTCTCTTCAGGAGTCAGGT 13 14 3.7 GACTCCTG GCAC HBB GCATGGTGCACCTTAACGGCAGACTTCTCCTCAGGA 13 19 5.2 GACTCCTG GTCAGGTGCAC HBB GCATGGTGCACCTACGGCAGACTTCTCCTCAGGAGT 13 17 5.3 GACTCCTG CAGGTGCAC HBB GCATGGTGCACCTGGCAGACTTCTCCTCAGGAGTCA 13 16 5.4 GACTCCTG GGTGCAC HBB GCATGGTGCACCTGCAGACTTCTCCTCAGGAGTCAG 13 13 5.5 GACTCCTG GTGCAC HBB GCATGGTGCACCTGACTTCTCCTCAGGAGTCAGGTG 13 12 5.6 GACTCCTG CAC HBB GCATGGTGCACCTACTTCTCCTCAGGAGTCAGGTGC 13 21 5.7 GACTCCTG AC HBB GCATGGTGCACCTTAACGGCAGACTTCTCCTCAGGA 12 19 5.8 GACTCCTG GTCAGGTGCA HBB GCATGGTGCACCTACGGCAGACTTCTCCTCAGGAGT 12 17 5.9 GACTCCTG CAGGTGCA HBB GCATGGTGCACCTGGCAGACTTCTCCTCAGGAGTCA 12 16 5.10 GACTCCTG GGTGCA HBB GCATGGTGCACCTGCAGACTTCTCCTCAGGAGTCAG 12 13 5.11 GACTCCTG GTGCA HBB GCATGGTGCACCTGACTTCTCCTCAGGAGTCAGGTG 12 12 5.12 GACTCCTG CA HBB GCATGGTGCACCTACTTCTCCTCAGGAGTCAGGTGC 12 14 5.13 GACTCCTG A HEXAS ATCCTTCCAGTCAATATCTTATGGCCCTGACTGGAA 13 14 1 GGGCCAT HEXAS ATCCTTCCAGTCATATATCTTATGGCCCTGACTGGAA 13 15 2 GGGCCAT HEXAS ATCCTTCCAGTCAGTATATCTTATGGCCCTGACTGGA 13 16 3 GGGCCAT A HEXAS ATCCTTCCAGTCAACCGTATATCTTATGGCCCTGACT 13 19 4 GGGCCAT GGAA HEXAS ATCCTTCCAGTCAAACCGTATATCTTATGGCCCTGAC 13 20 5 GGGCCAT TGGAA HEXAS ATCCTTCCAGTCAGAACCGTATATCTTATGGCCCTGA 13 21 6 GGGCCAT CTGGAA HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 13 22 7 GGGCCAT ACTGGAA HEXAS ATCCTTCCAGTCAATATCTTATGGCCCTGACT 9 14 8 GGGCCAT HEXAS ATCCTTCCAGTCATATATCTTATGGCCCTGACT 9 15 9 GGGCCAT HEXAS ATCCTTCCAGTCAGTATATCTTATGGCCCTGACT 9 16 10 GGGCCAT HEXAS ATCCTTCCAGTCAACCGTATATCTTATGGCCCTGACT 9 19 11 GGGCCAT HEXAS ATCCTTCCAGTCAAACCGTATATCTTATGGCCCTGAC 9 20 12 GGGCCAT T HEXAS ATCCTTCCAGTCAGAACCGTATATCTTATGGCCCTGA 9 21 13 GGGCCAT CT HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 9 22 14 GGGCCAT ACT HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 8 22 15 GGGCCAT AC HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 10 22 16 GGGCCAT ACTG HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 11 22 17 GGGCCAT ACTGG HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 12 22 18 GGGCCAT ACTGGA HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 13 22 19 GGGCCAT ACTGGAA HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 14 22 20 GGGCCAT ACTGGAAG HEXAS ATCCTTCCAGTCATGAACCGTATATCTTATGGCCCTG 15 22 21 GGGCCAT ACTGGAAGG HEXAS ATCCTTCCAGTCAACCTGAACCGTATATCTTATGGCC 9 25 22 GGGCCAT CTGACT HEXAS ATCCTTCCAGTCATACCTGAACCGTATATCTTATGGC 9 26 23 GGGCCAT CCTGACT HEXAS ATCCTTCCAGTCAGTACCTGAACCGTATATCTTATGG 9 27 24 GGGCCAT CCCTGACT HEXAS ATCCTTCCAGTCAGGTACCTGAACCGTATATCTTATG 9 28 25 GGGCCAT GCCCTGACT HEXAS ATCCTTCCAGTCATGGTACCTGAACCGTATATCTTAT 9 29 26 GGGCCAT GGCCCTGACT HEXA ATCCTTCCAGTCAACCTGAACCGTATATCCTATGGCC 13 21 5 GGGCCAT CTGACTGGAA HEXA ATCCTTCCAGTCAACCGTATATCCTATGGCCCTGACT 13 15 6 GGGCCAT GGAA HEXA ATCCTTCCAGTCAACCTGAACCGTATATCCTATGGCC 15 21 7 GGGCCAT CTGACTGGAAGG HEXA ATCCTTCCAGTCAACCTGAACCGTATATCCTATGGCC 14 21 8 GGGCCAT CTGACTGGAAG HEXA ATCCTTCCAGTCAACCTGAACCGTATATCCTATGGCC 12 21 9 GGGCCAT CTGACTGGA HEXA ATCCTTCCAGTCAACCTGAACCGTATATCCTATGGCC 11 21 10 GGGCCAT CTGACTGG HEXA ATCCTTCCAGTCAACCTGAACCGTATATCCTATGGCC 10 21 11 GGGCCAT CTGACTG HEXA ATCCTTCCAGTCAAACCGTATATCCTATGGCCCTGAC 13 16 12 GGGCCAT TGGAA HEXA ATCCTTCCAGTCATGAACCGTATATCCTATGGCCCTG 13 18 13 GGGCCAT ACTGGAA HEXA ATCCTTCCAGTCATACCTGAACCGTATATCCTATGGC 13 22 14 GGGCCAT CCTGACTGGAA HEXA ATCCTTCCAGTCATGGTACCTGAACCGTATATCCTAT 13 25 15 GGGCCAT GGCCCTGACTGGAA HEXAATCCTTCCAGTCA GTACCTGAACCGTATATCCTATGG 13 23 16 GGGCCAT CCCTGACTGGAAHEXA ATCCTTCCAGTCA AACCGTATATCCTATGGCCCTGAC 10 16 17 GGGCCAT TG HEXAATCCTTCCAGTCA TGAACCGTATATCCTATGGCCCTG 10 18 18 GGGCCAT ACTG HEXAATCCTTCCAGTCA TACCTGAACCGTATATCCTATGGC 10 22 19 GGGCCAT CCTGACTG HEXAATCCTTCCAGTCA TGGTACCTGAACCGTATATCCTAT 10 25 20 GGGCCAT GGCCCTGACTG

TABLE 3Q FIGs. 50A-50B nicking sgRNA NICKING SGRNA SPACER SEQUENCEHBB_ED7A_+72 GCCTTGATACCAACCTGCCCA (SEQ ID NO: 626) HEXA_ED7B_+60GCTGGAACTGGTCACCAAGGC (SEQ ID NO: 627) HEXA_ED7B_CORRECT_WT_PE3BGTACCTGAACCGTATATCCTA (SEQ ID NO: 628) HEXA_ED7B_CORRECT_SILENT_PE3BGTACCTGAACCGTATATCTTA (SEQ ID NO: 629)

TABLE 3R FIGs. 51A-51G PEgRNA RT PBS TEMPLATE SPACER SEQUENCE3' EXTENSION (SEQ LENGTH LENGTH PEGRNA (SEQ ID NO: 632-640)ID NO: 641-649) (NT) (NT) HEK3_ED8_1TTOG GGCCCAGACTGAGCTCTGCCATCCCGTGCTCA 13 10 ACGTGA GTCTG HEK3_ED8_3ATOC GGCCCAGACTGAGCTCTGCCAGCACGTGCTCA 13 10 ACGTGA GTCTG HEK3_ED8_3ATOT GGCCCAGACTGAGCTCTGCCAACACGTGCTCA 13 10 ACGTGA GTCTG HEK3_ED8_3 GGCCCAGACTGAGCTGGAGGAAGCAGGGCTTC 13 34 ATOT_5- ACGTGA CTTTCCTCTGAAAACACG 6GGTOTTTGCTCAGTCTG HEK3_ED8_1CTTINS GGCCCAGACTGAGC TCTGCCATCAAAGCGTGC 13 10ACGTGA TCAGTCTG RNF2_ED8_1CTOA GTCATCTTAGTCATT AACGAACACCTCATGTAA 15 14ACCTG TGACTAAGATG RNF2_ED8_1CTOG GTCATCTTAGTCATT AACGAACACCTCACGTAA 1514 ACCTG TGACTAAGATG RNF2_ED8_1GTAINS GTCATCTTAGTCATT AACGAACACCTCAGTACG15 17 ACCTG TAATGACTAAGATG HBB_ED8_4ATOT GCATGGTGCACCTGAGACTTCTCCACAGGAGT 13 14 ACTCCTG CAGGTGCAC

TABLE 4 Sequences of primers used for mammalian cell genomicDNA amplification and HTS¹⁸¹. DESCRIPTIONSEQUENCE (SEQ ID NOS: 3811-3863) HEK3 FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGTG GGCTGCCTAGAAAGG HEK3 REVTGGAGTTCAGACGTGTGCTCTTCCGATCTCCCAGCCAAACTT GTCAACC RNF2 FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACGTC TCATATGCCCCTTGG RNF2 REVTGGAGTTCAGACGTGTGCTCTTCCGATCTACGTAGGAATTTT GGTGGGACA HEK4 FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAACC CAGGTAGCCAGAGAC HEK4 REVTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTTCAACCCG AACGGAG EMX1 FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGCT CAGCCTGAGTGTTGA EMX1 REVTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTGGGTTTGT GGTTGC FANCF FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCATTG CAGAGAGGCGTATCA FANCF REVTGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGTCCCAGGT GCTGAC HBB FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGGGT TGGCCAATCTACTCCC HBB REVTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTTCTCTGTCT CCACATGCC PRNP FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCAG TGGAACAAGCCGAGT PRNP REVTGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGGTTGGGGT AACGGTG HEXA FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCATAC AGGTGTGGCGAGAGG HEXA REVTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGCCTCCTTTG GTTAGCA RUNX1 FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCACA AACAAGACAGGGAACTG RUNX1 REVTGGAGTTCAGACGTGTGCTCTTCCGATCTAGATGTAGGGCTA GAGGGGTG VEGFA FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACTTG GTGCCAAATTCTTCTCC VEGFA REVTGGAGTTCAGACGTGTGCTCTTCCGATCTAAAGAGGGAATG GGCTTTGGA DNMT FWDACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACAA CAGCTTCATGTCAGCC DNMT REVTGGAGTTCAGACGTGTGCTCTTCCGATCTACGTTAATGTTTC CTGATGGTCC HEK3 OFF-TARGETACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCCC SITE 1 FWD TGTTGACCTGGAGAAHEK3 OFF-TARGET TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGTACTTGCC SITE 1 REVCTGACCA HEK3 OFF-TARGET ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTGGTSITE 2 FWD GTTGACAGGGAGCAA HEK3 OFF-TARGETTGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGATGTGGGC SITE 2 REV AGAAGGGHEK3 OFF-TARGET ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAGA SITE 3 FWDGGGAACAGAAGGGCT HEK3 OFF-TARGETTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCAAAGGCCC SITE 3 REV AAGAACCTHEK3 OFF-TARGET ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTA SITE 4 FWDGCACTTTGGAAGGTCG HEK3 OFF-TARGETTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTCATCTTAATCT SITE 4 REV GCTCAGCCHEK4 OFF-TARGET ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCAT SITE 1 FWDGGCTTCTGAGACTCA HEK4 OFF-TARGETTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCCCTTGCAC SITE 1 REV TCCCTGTCTTTHEK4 OFF-TARGET ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTGG SITE 2 FWDCAATGGAGGCATTGG HEK4 OFF-TARGETTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGAGGCTGCC SITE 2 REV CATGAGAGHEK4 OFF-TARGET ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTCT SITE 3 FWDGAGGCTCGAATCCTG HEK4 OFF-TARGETTGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTGGCCTCCAT SITE 3 REV ATCCCTGHEK4 OFF-TARGET ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTCC SITE 4 FWDACCAGAACTCAGCCC HEK4 OFF-TARGETTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCGGTTCCTCC SITE 4 REV ACAACAC EMX1 OFF-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGGG TARGET SITE 1GAGATTTGCATCTGTGGAGG FWD EMX1 OFF-TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTTTATACCATC TARGET SITE 1 TTGGGGTTACAGREV EMX1 OFF- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAATG TARGET SITE 2TGCTTCAACCCATCACGGC FWD EMX1 OFF-TGGAGTTCAGACGTGTGCTCTTCCGATCTCCATGAATTTGTG TARGET SITE 2 ATGGATGCAGTCTGREV EMX1 OFF- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGAA TARGET SITE 3GGAGGTGCAGGAGCTAGAC FWD EMX1 OFF-TGGAGTTCAGACGTGTGCTCTTCCGATCTCATCCCGACCTTC TARGET SITE 3 ATCCCTCCTGG REVEMX1 OFF- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTAGT TARGET SITE 4TCTGACATTCCTCCTGAGGG FWD EMX1 OFF-TGGAGTTCAGACGTGTGCTCTTCCGATCTTCAAACAAGGTG TARGET SITE 4 CAGATACAGCA REVFANCF OFF- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCGGG TARGET SITE 1CAGTGGCGTCTTAGTCG FWD FANCF OFF-TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCTGGGTTTGGT TARGET SITE 1 TGGCTGCTC REVFANCF OFF- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTCCT TARGET SITE 2TGCCGCCCAGCCGGTC FWD FANCF OFF-TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGGGGAAGA TARGET SITE 2 GGCGAGGACAC REVFANCF OFF- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGT TARGET SITE 3GTTTCCCATCCCCAACAC FWD FANCF OFF-TGGAGTTCAGACGTGTGCTCTTCCGATCTGAATGGATCCCCC TARGET SITE 3 CCTAGAGCTC REVFANCF OFF- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGGC TARGET SITE 4CCACAGGTCCTTCTGGA FWD

TABLE 5 Sequences of 100-mer single-stranded DNA oligonucleotide donortemplates used in HDR experiments and in the creation of the HBBE6V HEK293T cell line. Oligonucleotides are 100-103 nt in length withhomology arms centered around the site of the edit. Oligonucleotideswere from Integrated DNA Technologies, purified by PAGE. HEK3 +3 A TO TGCTTCTCCAGCCCTGGCCTGGGTCAATCCTTGGGGCCCAGACTGAGCACGTGTTGGCAGAGGAAAGGAAGCCCTGCTTCCTCCAGAGGGCGTCGCAGGAC (SEQ ID NO: 717) HEK3 +3 A TO T, +5, 6GCTTCTCCAGCCCTGGCCTGGGTCAATCCTTGGGGCCCAG GGTOTTACTGAGCACGTGTTTTCAGAGGAAAGGAAGCCCTGCTTCCTCCAGAGGGCGTCGCAGGAC (SEQ ID NO: 718) HEK3 +1 T TO GGCTTCTCCAGCCCTGGCCTGGGTCAATCCTTGGGGCCCAGACTGAGCACGGGATGGCAGAGGAAAGGAAGCCCTGCTTCCTCCAGAGGGCGTCGCAGGAC (SEQ ID NO: 719) HEK3 +3 A TO CGCTTCTCCAGCCCTGGCCTGGGTCAATCCTTGGGGCCCAGACTGAGCACGTGCTGGCAGAGGAAAGGAAGCCCTGCTTCCTCCAGAGGGCGTCGCAGGAC (SEQ ID NO: 720) HEK3 +1 CTTGCTTCTCCAGCCCTGGCCTGGGTCAATCCTTGGGGCCCAG INSERTIONACTGAGCACGCTTTGATGGCAGAGGAAAGGAAGCCCTGCTTCCTCCAGAGGGCGTCGCAGGAC (SEQ ID NO: 721) RNF2 +1 C TO ACCCAGTTTACACGTCTCATATGCCCCTTGGCAGTCATCTTAGTCATTACATGAGGTGTTCGTTGTAACTCATATAAACTGAGTTCCCATGTTTTGCTTAA (SEQ ID NO: 722) RNF2 +1 C TO GCCCAGTTTACACGTCTCATATGCCCCTTGGCAGTCATCTTAGTCATTACGTGAGGTGTTCGTTGTAACTCATATAAACTGAGTTCCCATGTTTTGCTTAA (SEQ ID NO: 723) RNF2 +1 GTACAGTTTACACGTCTCATATGCCCCTTGGCAGTCATCTTAGT INSERTIONCATTACGTACTGAGGTGTTCGTTGTAACTCATATAAACTGAGTTCCCATGTTTTGCTTA (SEQ ID NO: 724) HBB E6VACTTCATCCACGTTCACCTTGCCCCACAGGGCAGTAACGG INSTALLATION (ALSOCAGACTTCTCCACAGGAGTCAGATGCACCATGGTGTCTGT USED FOR CREATIONTTGAGGTTGCTAGTGAACAC (SEQ ID NO: 725) OF THE HBB E6V HEK293T CELL LINE)HBB E6V ACTTCATCCACGTTCACCTTGCCCCACAGGGCAGTAACGG CORRECTIONCAGACTTCTCCTCAGGAGTCAGGTGCACCATGGTGTCTGT PROTOSPACER ATTGAGGTTGCTAGTGAACAC (SEQ ID NO: 726) HBB E6VGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCACCT CORRECTIONGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGC PROTOSPACER BAAGGTGAACGTGGATGAAGT (SEQ ID NO: 727) HBB E6VGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCACCT CORRECTIONGACTCCTGATGAGAAGTCTGCCGTTACTGCCCTGTGGGGC PROTOSPACER B,AAGGTGAACGTGGATGAAGT (SEQ ID NO: 728) SILENT PAM MUTATION PRNP G127VCACATGGCTGGTGCTGCAGCAGCTGGGGCAGTGGTGGGGGGCCTTGGCGTCTACATGCTGGGAAGTGCCATGAGCAGGCCCATCATACATTTCGGCAGTG (SEQ ID NO: 729)

Additional Sequences

Sequences of yeast dual fluorescent reporter plasmids used herein

p425-GFP_stop_mCherry: (SEQ ID NO: 730)ATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGATCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAACTATTTTTTTCAAAGATGACGGTAACTACAAGACCAGAGCTGAAGTCAAGTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGAAGATGGTAACATTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTACATCATGGCTGACAAACAAAAGAATGGTATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATTGGTGATGGTCCAGTCTTGTTACCAGACAACCATTACTTATCCACTCAATCTGCCTTATCCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTACTGCTGCTGGTATTACCCATGGTATGGATGAATTGTACAAAGCTAGCAACCTGGGTCAA TCCTTGGGGCCCAGACTGAGCACGTGA TGGCAGAGCACAGGAGACGTCATGGTTTCAAAAGGTGAAGAAGATAATATGGCTATTATTAAAGAATTTATGAGATTTAAAGTTCATATGGAAGGTTCAGTTAATGGTCATGAATTTGAAATTGAAGGTGAAGGTGAAGGTAGACCATATGAAGGTACTCAAACTGCTAAATTGAAAGTTACTAAAGGTGGTCCATTACCATTTGCTTGGGATATTTTGTCACCACAATTTATGTATGGTTCAAAAGCTTATGTTAAACATCCAGCTGATATTCCAGATTATTTAAAATTGTCATTTCCAGAAGGTTTTAAATGGGAAAGAGTTATGAATTTTGAAGATGGTGGTGTTGTTACTGTTACTCAAGATTCATCATTACAAGATGGTGAATTTATTTATAAAGTTAAATTGAGAGGTACTAATTTTCCATCAGATGGTCCAGTTATGCAAAAAAAAACTATGGGTTGGGAAGCTTCATCAGAAAGAATGTATCCAGAAGATGGTGCTTTAAAAGGTGAAATTAAACAAAGATTGAAATTAAAAGATGGTGGTCATTATGATGCTGAAGTTAAAACTACTTATAAAGCTAAAAAACCAGTTCAATTACCAGGTGCTTATAATGTTAATATTAAATTGGATATTACTTCACATAATGAAGATTATACTATTGTTGAACAATATGAAAGAGCTGAAGGTAGACATTCAACTGGTGGTATGGATGAATTATATAAAGGTACCGCTCGAGCAGCTGTGATTGATTGAGTCGACTTGGTTGAACACGTTGCCAAGGCTTAAGTGAATTTACTTTAAATCTTGCATTTAAATAAATTTTCTTTTTATAGCTTTATGACTTAGTTTCAATTTATATACTATTTTAATGACATTTTCGATTCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATCGACTACGTCGTAAGGCCGTTTCTGACAGAGTAAAATTCTTGAGGGAACTTTCACCATTATGGGAAATGCTTCAAGAAGGTATTGACTTAAACTCCATCAAATGGTCAGGTCATTGAGTGTTTTTTATTTGTTGTATTTTTTTTTTTTTAGAGAAAATCCTCCAATATCAAATTAGGAATCGTAGTTTCATGATTTTCTGTTACACCTAACTTTTTGTGTGGTGCCCTCCTCCTTGTCAATATTAATGTTAAAGTGCAATTCTTTTTCCTTATCACGTTGAGCCATTAGTATCAATTTGCTTACCTGTATTCCTTTACTATCCTCCTTTTTCTCCTTCTTGATAAATGTATGTAGATTGCGTATATAGTTTCGTCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTCAACCTTATTCTTTGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAAGATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCGGTACCCCATTTAGGACCAGCCACAGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTAGCATCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATAGGGGCAGACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAAGCCGGTTCCGGCCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTAACTGCGGTCAAGATATTTCTTGAATCAGGCGCCTTAGACCGCTCGGCCAAACAACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAACGTTTTTGAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAATGTGGATTTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTATGTGGATATACTAGAAGTTCTCCTCGACCGTCGATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCGTTAGAATCATTTTGAATAAAAAACACGCTTTTTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGAATTC p425-GFP_+1fs_mCherry: (SEQ ID NO: 731)ATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGATCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAACTATTTTTTTCAAAGATGACGGTAACTACAAGACCAGAGCTGAAGTCAAGTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGAAGATGGTAACATTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTACATCATGGCTGACAAACAAAAGAATGGTATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATTGGTGATGGTCCAGTCTTGTTACCAGACAACCATTACTTATCCACTCAATCTGCCTTATCCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTACTGCTGCTGGTATTACCCATGGTATGGATGAATTGTACAAAGCTAGCAAACCTGGGTCA ATCCTTGGGGCCCAGACTGAGCACGTGA TGGCAGAGCACAGGAGACGTCATGGTTTCAAAAGGTGAAGAAGATAATATGGCTATTATTAAAGAATTTATGAGATTTAAAGTTCATATGGAAGGTTCAGTTAATGGTCATGAATTTGAAATTGAAGGTGAAGGTGAAGGTAGACCATATGAAGGTACTCAAACTGCTAAATTGAAAGTTACTAAAGGTGGTCCATTACCATTTGCTTGGGATATTTTGTCACCACAATTTATGTATGGTTCAAAAGCTTATGTTAAACATCCAGCTGATATTCCAGATTATTTAAAATTGTCATTTCCAGAAGGTTTTAAATGGGAAAGAGTTATGAATTTTGAAGATGGTGGTGTTGTTACTGTTACTCAAGATTCATCATTACAAGATGGTGAATTTATTTATAAAGTTAAATTGAGAGGTACTAATTTTCCATCAGATGGTCCAGTTATGCAAAAAAAAACTATGGGTTGGGAAGCTTCATCAGAAAGAATGTATCCAGAAGATGGTGCTTTAAAAGGTGAAATTAAACAAAGATTGAAATTAAAAGATGGTGGTCATTATGATGCTGAAGTTAAAACTACTTATAAAGCTAAAAAACCAGTTCAATTACCAGGTGCTTATAATGTTAATATTAAATTGGATATTACTTCACATAATGAAGATTATACTATTGTTGAACAATATGAAAGAGCTGAAGGTAGACATTCAACTGGTGGTATGGATGAATTATATAAAGGTACCGCTCGAGCAGCTGTGATTGATTGAGTCGACTTGGTTGAACACGTTGCCAAGGCTTAAGTGAATTTACTTTAAATCTTGCATTTAAATAAATTTTCTTTTTATAGCTTTATGACTTAGTTTCAATTTATATACTATTTTAATGACATTTTCGATTCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATCGACTACGTCGTAAGGCCGTTTCTGACAGAGTAAAATTCTTGAGGGAACTTTCACCATTATGGGAAATGCTTCAAGAAGGTATTGACTTAAACTCCATCAAATGGTCAGGTCATTGAGTGTTTTTTATTTGTTGTATTTTTTTTTTTTTAGAGAAAATCCTCCAATATCAAATTAGGAATCGTAGTTTCATGATTTTCTGTTACACCTAACTTTTTGTGTGGTGCCCTCCTCCTTGTCAATATTAATGTTAAAGTGCAATTCTTTTTCCTTATCACGTTGAGCCATTAGTATCAATTTGCTTACCTGTATTCCTTTACTATCCTCCTTTTTCTCCTTCTTGATAAATGTATGTAGATTGCGTATATAGTTTCGTCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTCAACCTTATTCTTTGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAAGATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCGGTACCCCATTTAGGACCAGCCACAGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTAGCATCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATAGGGGCAGACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAAGCCGGTTCCGGCCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTAACTGCGGTCAAGATATTTCTTGAATCAGGCGCCTTAGACCGCTCGGCCAAACAACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAACGTTTTTGAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAATGTGGATTTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTATGTGGATATACTAGAAGTTCTCCTCGACCGTCGATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCGTTAGAATCATTTTGAATAAAAAACACGCTTTTTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGAATTC p425-GFP_−1fs_mCherry: (SEQ ID NO: 732)ATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGATCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAACTATTTTTTTCAAAGATGACGGTAACTACAAGACCAGAGCTGAAGTCAAGTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGAAGATGGTAACATTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTACATCATGGCTGACAAACAAAAGAATGGTATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATTGGTGATGGTCCAGTCTTGTTACCAGACAACCATTACTTATCCACTCAATCTGCCTTATCCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTACTGCTGCTGGTATTACCCATGGTATGGATGAATTGTACAAAGCTAGCAAACCTGGGTCA ATCCTTGGGGCCCAGACTGAGCACGTG A TGGCAGAGCACAGGACGTCATGGTTTCAAAAGGTGAAGAAGATAATATGGCTATTATTAAAGAATTTATGAGATTTAAAGTTCATATGGAAGGTTCAGTTAATGGTCATGAATTTGAAATTGAAGGTGAAGGTGAAGGTAGACCATATGAAGGTACTCAAACTGCTAAATTGAAAGTTACTAAAGGTGGTCCATTACCATTTGCTTGGGATATTTTGTCACCACAATTTATGTATGGTTCAAAAGCTTATGTTAAACATCCAGCTGATATTCCAGATTATTTAAAATTGTCATTTCCAGAAGGTITTAAATGGGAAAGAGTTATGAATTTTGAAGATGGTGGTGTTGTTACTGTTACTCAAGATTCATCATTACAAGATGGTGAATTTATTTATAAAGTTAAATTGAGAGGTACTAATTTTCCATCAGATGGTCCAGTTATGCAAAAAAAAACTATGGGTTGGGAAGCTTCATCAGAAAGAATGTATCCAGAAGATGGTGCTTTAAAAGGTGAAATTAAACAAAGATTGAAATTAAAAGATGGTGGTCATTATGATGCTGAAGTTAAAACTACTTATAAAGCTAAAAAACCAGTTCAATTACCAGGTGCTTATAATGTTAATATTAAATTGGATATTACTTCACATAATGAAGATTATACTATTGTTGAACAATATGAAAGAGCTGAAGGTAGACATTCAACTGGTGGTATGGATGAATTATATAAAGGTACCGCTCGAGCAGCTGTGATTGATTGAGTCGACTTGGTTGAACACGTTGCCAAGGCTTAAGTGAATTTACTTTAAATCTTGCATTTAAATAAATTTTCTTTTTATAGCTTTATGACTTAGTTTCAATTTATATACTATTTTAATGACATTTTCGATTCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATCGACTACGTCGTAAGGCCGTTTCTGACAGAGTAAAATTCTTGAGGGAACTTTCACCATTATGGGAAATGCTTCAAGAAGGTATTGACTTAAACTCCATCAAATGGTCAGGTCATTGAGTGTTTTTTATTTGTTGTATTTTTTTTTTTTTAGAGAAAATCCTCCAATATCAAATTAGGAATCGTAGTTTCATGATTTTCTGTTACACCTAACTTTTTGTGTGGTGCCCTCCTCCTTGTCAATATTAATGTTAAAGTGCAATTCTTTTTCCTTATCACGTTGAGCCATTAGTATCAATTTGCTTACCTGTATTCCTTTACTATCCTCCTTTTTCTCCTTCTTGATAAATGTATGTAGATTGCGTATATAGTTTCGTCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTCAACCTTATTCTTTGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAAGATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCGGTACCCCATTTAGGACCAGCCACAGCACCTAACAAAACGGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTAGCATCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATAGGGGCAGACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAAGCCGGTTCCGGCCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTAACTGCGGTCAAGATATTTCTTGAATCAGGCGCCTTAGACCGCTCGGCCAAACAACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAACGTTTTTGAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAATGTGGATTTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTATGTGGATATACTAGAAGTTCTCCTCGACCGTCGATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCGTTAGAATCATTTTGAATAAAAAACACGCTTTTTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGAATTC KEY: GFP open reading frameLinker containing stop codon +1 frameshift, or −1 frameshiftmCherry open reading framePlasmid backbone (containing the GPD promoter, Leu2 marker, and AmpR)Protospacer (underlined) PAM (boldfaced)

DNA sequences of mammalian prime editor plasmids and example PEgRNAplasmid

pCMV-PE1: (SEQ ID NO: 733)ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGTGACTCTGGAGGATCTAGCGGAGGATCCTCTGGCAGCGAGACACCAGGAACAAGCGAGTCAGCAACACCAGAGAGCAGTGGCGGCAGCAGCGGCGGC AGCAGCACCCTAAATATAGAAGATGAGTATCGGCTACATGAGACCTCAAAAGAGCCAGATGTTTCTCTAGGGTCCACATGGCTGTCTGATTTTCCTCAGGCCTGGGCGGAAACCGGGGGCATGGGACTGGCAGTTCGCCAAGCTCCTCTGATCATACCTCTGAAAGCAACCTCTACCCCCGTGTCCATAAAACAATACCCCATGTCACAAGAAGCCAGACTGGGGATCAAGCCCCACATACAGAGACTGTTGGACCAGGGAATACTGGTACCCTGCCAGTCCCCCTGGAACACGCCCCTGCTACCCGTTAAGAAACCAGGGACTAATGATTATAGGCCTGTCCAGGATCTGAGAGAAGTCAACAAGCGGGTGGAAGACATCCACCCCACCGTGCCCAACCCTTACAACCTCTTGAGCGGGCTCCCACCGTCCCACCAGTGGTACACTGTGCTTGATTTAAAGGATGCCTTTTTCTGCCTGAGACTCCACCCCACCAGTCAGCCTCTCTTCGCCTTTGAGTGGAGAGATCCAGAGATGGGAATCTCAGGACAATTGACCTGGACCAGACTCCCACAGGGTTTCAAAAACAGTCCCACCCTGTTTGATGAGGCACTGCACAGAGACCTAGCAGACTTCCGGATCCAGCACCCAGACTTGATCCTGCTACAGTACGTGGATGACTTACTGCTGGCCGCCACTTCTGAGCTAGACTGCCAACAAGGTACTCGGGCCCTGTTACAAACCCTAGGGAACCTCGGGTATCGGGCCTCGGCCAAGAAAGCCCAAATTTGCCAGAAACAGGTCAAGTATCTGGGGTATCTTCTAAAAGAGGGTCAGAGATGGCTGACTGAGGCCAGAAAAGAGACTGTGATGGGGCAGCCTACTCCGAAGACCCCTCGACAACTAAGGGAGTTCCTAGGGACGGCAGGCTTCTGTCGCCTCTGGATCCCTGGGTTTGCAGAAATGGCAGCCCCCCTGTACCCTCTCACCAAAACGGGGACTCTGTTTAATTGGGGCCCAGACCAACAAAAGGCCTATCAAGAAATCAAGCAAGCTCTTCTAACTGCCCCAGCCCTGGGGTTGCCAGATTTGACTAAGCCCTTTGAACTCTTTGTCGACGAGAAGCAGGGCTACGCCAAAGGTGTCCTAACGCAAAAACTGGGACCTTGGCGTCGGCCGGTGGCCTACCTGTCCAAAAAGCTAGACCCAGTAGCAGCTGGGTGGCCCCCTTGCCTACGGATGGTAGCAGCCATTGCCGTACTGACAAAGGATGCAGGCAAGCTAACCATGGGACAGCCACTAGTCATTCTGGCCCCCCATGCAGTAGAGGCACTAGTCAAACAACCCCCCGACCGCTGGCTTTCCAACGCCCGGATGACTCACTATCAGGCCTTGCTTTTGGACACGGACCGGGTCCAGTTCGGACCGGTGGTAGCCCTGAACCCGGCTACGCTGCTCCCACTGCCTGAGGAAGGGCTGCAACACAACTGCCTTGATATCCTGGCCGAAGCCCACGGAACCCGACCCGACCTAACGGACCAGCCGCTCCCAGACGCCGACCACACCTGGTACACGGATGGAAGCAGTCTCTTACAAGAGGGACAGCGTAAGGCGGGAGCTGCGGTGACCACCGAGACCGAGGTAATCTGGGCTAAAGCCCTGCCAGCCGGGACATCCGCTCAGCGGGCTGAACTGATAGCACTCACCCAGGCCCTAAAGATGGCAGAAGGTAAGAAGCTAAATGTTTATACTGATAGCCGTTATGCTTTTGCTACTGCCCATATCCATGGAGAAATATACAGAAGGCGTGGGTTGCTCACATCAGAAGGCAAAGAGATCAAAAATAAAGACGAGATCTTGGCCCTACTAAAAGCCCTCTTTCTGCCCAAAAGACTTAGCATAATCCATTGTCCAGGACATCAAAAGGGACACAGCGCCGAGGCTAGAGGCAACCGGATGGCTGACCAAGCGGCCCGAAAGGCAGCCATCACAGAGACTCCAGACACCTCTACCCTCCTCATAGAAAATTCATCACCCTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTCTAA CCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGAAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACC pCMV-PE2: (SEQ ID NO: 734)ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGTGACTCTGGAGGATCTAGCGGAGGATCCTCTGGCAGCGAGACACCAGGAACAAGCGAGTCAGCAACACCAGAGAGCAGTGGCGGCAGCAGCGGCGGCAGCAGCACCCTAAATATAGAAGATGAGTATCGGCTACATGAGACCTCAAAAGAGCCAGATGTTTCTCTAGGGTCCACATGGCTGTCTGATTTTCCTCAGGCCTGGGCGGAAACCGGGGGCATGGGACTGGCAGTTCGCCAAGCTCCTCTGATCATACCTCTGAAAGCAACCTCTACCCCCGTGTCCATAAAACAATACCCCATGTCACAAGAAGCCAGACTGGGGATCAAGCCCCACATACAGAGACTGTTGGACCAGGGAATACTGGTACCCTGCCAGTCCCCCTGGAACACGCCCCTGCTACCCGTTAAGAAACCAGGGACTAATGATTATAGGCCTGTCCAGGATCTGAGAGAAGTCAACAAGCGGGTGGAAGACATCCACCCCACCGTGCCCAACCCTTACAACCTCTTGAGCGGGCTCCCACCGTCCCACCAGTGGTACACTGTGCTTGATTTAAAGGATGCCTTTTTCTGCCTGAGACTCCACCCCACCAGTCAGCCTCTCTTCGCCTTTGAGTGGAGAGATCCAGAGATGGGAATCTCAGGACAATTGACCTGGACCAGACTCCCACAGGGTTTCAAAAACAGTCCCACCCTGTTTAATGAGGCACTGCACAGAGACCTAGCAGACTTCCGGATCCAGCACCCAGACTTGATCCTGCTACAGTACGTGGATGACTTACTGCTGGCCGCCACTTCTGAGCTAGACTGCCAACAAGGTACTCGGGCCCTGTTACAAACCCTAGGGAACCTCGGGTATCGGGCCTCGGCCAAGAAAGCCCAAATTTGCCAGAAACAGGTCAAGTATCTGGGGTATCTTCTAAAAGAGGGTCAGAGATGGCTGACTGAGGCCAGAAAAGAGACTGTGATGGGGCAGCCTACTCCGAAGACCCCTCGACAACTAAGGGAGTTCCTAGGGAAGGCAGGCTTCTGTCGCCTCTTCATCCCTGGGTTTGCAGAAATGGCAGCCCCCCTGTACCCTCTCACCAAACCGGGGACTCTGTTTAATTGGGGCCCAGACCAACAAAAGGCCTATCAAGAAATCAAGCAAGCTCTTCTAACTGCCCCAGCCCTGGGGTTGCCAGATTTGACTAAGCCCTTTGAACTCTTTGTCGACGAGAAGCAGGGCTACGCCAAAGGTGTCCTAACGCAAAAACTGGGACCTTGGCGTCGGCCGGTGGCCTACCTGTCCAAAAAGCTAGACCCAGTAGCAGCTGGGTGGCCCCCTTGCCTACGGATGGTAGCAGCCATTGCCGTACTGACAAAGGATGCAGGCAAGCTAACCATGGGACAGCCACTAGTCATTCTGGCCCCCCATGCAGTAGAGGCACTAGTCAAACAACCCCCCGACCGCTGGCTTTCCAACGCCCGGATGACTCACTATCAGGCCTTGCTTTTGGACACGGACCGGGTCCAGTTCGGACCGGTGGTAGCCCTGAACCCGGCTACGCTGCTCCCACTGCCTGAGGAAGGGCTGCAACACAACTGCCTTGATATCCTGGCCGAAGCCCACGGAACCCGACCCGACCTAACGGACCAGCCGCTCCCAGACGCCGACCACACCTGGTACACGGATGGAAGCAGTCTCTTACAAGAGGGACAGCGTAAGGCGGGAGCTGCGGTGACCACCGAGACCGAGGTAATCTGGGCTAAAGCCCTGCCAGCCGGGACATCCGCTCAGCGGGCTGAACTGATAGCACTCACCCAGGCCCTAAAGATGGCAGAAGGTAAGAAGCTAAATGTTTATACTGATAGCCGTTATGCTTTTGCTACTGCCCATATCCATGGAGAAATATACAGAAGGCGTGGGTGGCTCACATCAGAAGGCAAAGAGATCAAAAATAAAGACGAGATCTTGGCCCTACTAAAAGCCCTCTTTCTGCCCAAAAGACTTAGCATAATCCATTGTCCAGGACATCAAAAGGGACACAGCGCCGAGGCTAGAGGCAACCGGATGGCTGACCAAGCGGCCCGAAAGGCAGCCATCACAGAGACTCCAGACACCTCTACCCTCCTCATAGAAAATTCATCACCCTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGAAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAG GGAGAGCCGCCACCN-terminal NLS + Cas9 H840A Flexible linkerM-MLV reverse transcriptase + C-terminal NLSPlasmid backbone (containing CMV promoter and AmpR)pU6-HEK3_PEgRNA_CTTins: (SEQ ID NO: 735)GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAG TCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTG TTTTTTTAAGCTTGGGCCGCTCGAGGTACCTCTCTACATATGACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGCTAGCTGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAA U6 Promoter sequence Spacer sequence sgRNA scaffold3 ′  extension (contains PBS and RT template) Backbone (contains AmpR)pLenti-hSyn-N-PE2-NpuN-P2A-GFP-KASH_U6-DNMT1-PEgRNA: (SEQ ID NO: 736)GTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGCGCGTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGTTAATTAAGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC GCGGGCTGGAGCTGTTCGCGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGATGCAAGCGC GAACAGCTCCAGTTTTTTTGAATTC AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG

ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAG TGCCTGTCCTACGAGACAGAGATCCTGACAGTGGAGTATGGCCTGCTGCCAATCGGCAAGATCGTGGAGAAGAGGATCGAGTGTACCGTGTACTCTGTGGATAACAATGGCAACATCTATACACAGCCCGTGGCACAGTGGCACGATAGGGGAGAGCAGGAGGTGTTCGAGTATTGCCTGGAGGACGGCAGCCTGATCAGGGCAACCAAGGACCACAAGTTCATGACAGTGGATGGCCAGATGCTGCCCATCGACGAGATTTTCGAGCGGGAGCTGGACCTGATGAGAGTGGATAACCTGCCTAAT

ACGCGTTAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGTCGACTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGT GCCACCTGACU6promoter PEgRNA hSynpromoter N-termPE2 N-termNpu

pLenti-hSyn-C-PE2-NpuC: (SEQ ID NO: 737)GTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGCGCGTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGTTAATTAAGGTACCAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG TCGAGAATCTAGAGCGCTGCCACCATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTC ATCAAGATTGCTACACGGAAATACCTGGGAAAGCAGAACGTGTACGACATCGGCGTGGAGCGGGATCACAACTTCGCCCTGAAGAATGGCTTTATCGCCAGCAAT TGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTCTAA ACGCGTTAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGTCGACTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC hSynpromoter C-termNpu C-termwtCas9pLenti-U6-DNMT1_nicking_sgRNA: (SEQ ID NO: 738)TAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCAGAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC

TTTTTTAAGCTTGGCGTAACTAGATCTTGAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCACTTTGGCGCCGGCTCGAGGGGGCCCGGGTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACGTACGGCCACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCCGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGAGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGAACGCGTTAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGTCGACTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTACGTATAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACA U6promoter

Amino acid sequences of Moloney murine leukemia virus reversetranscriptase (M-MLV RT) variants used herein.

PE1 M-MLV RT: (SEQ ID NO: 739)TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPM3 M-MLV RT (D200N, T330P, L603W) (see Baranauskas et al.¹⁸²):(SEQ ID NO: 740)TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPPE2 M-MLV RT (D200N, T306K, W313F, T330P, L603W): (SEQ ID NO: 741)TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP M3-deadRT M-MLV RT:(SEQ ID NO: 742)TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKLPGTNDYSPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

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Each of the following references are cited in Example 12, each of whichare incorporated herein by reference.

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Example 13—Cell Data Recording and Lineage Tracing by PE Background

Genome modification can be used to study and record cellular processesand development. Linking cellular events, like cell division orsignaling cascade activation, to DNA sequence modifications storescellular histories as interpretable DNA sequence changes that woulddescribe whether specific cellular events had taken place. DNA editingis necessary for these applications because DNA is faithfully passedfrom one cell to the next in a way that RNA and proteins are not.Information relating to cellular state and lineage are in general lostwhen modifications are made to short-lived protein and RNA molecules.Recording cellular events within a single cell is a powerful way tounderstand how disease states are initiated, maintained, and changedrelative to healthy controls. The ability to probe these questions hasimplications for understanding the development of cancer, neurologicaldisease, and a host of other important problems in human health. primeediting (PE) provides a system for creating targeted andsequence-specified genomic insertions, deletions, or mutations. Repeatedmodification of a DNA target that can be sequenced by targeted ampliconsequencing and/or RNA sequencing (which is of particular value forsingle cell recording experiments) can be used to record a host ofimportant biological processes, including activation of signalingcascades, metabolic states, and cellular differentiation programs.Connecting internal and external cellular signals to sequencemodifications in the genome is, in theory, possible for any signal forwhich a signal responsive promoter exists. It is believed that PEenables a greatly expanded toolkit for probing the cellular lineage andthe signaling histories of eukaryotic and prokaryotic cells in bothcultured conditions and in vivo.

Previous Standards

Targeted sequence insertion, deletion, or mutation can be used to studya number of important biological questions including lineage tracing andrecording of cellular stimuli. The current toolkit for generating thesesignatures in the genome is limited. Mutagenesis of target loci has beendeveloped to date using both DNA-nucleases and base editors.

CRISPR/Cas9 nuclease cutting of target sequences generates stochasticsequence changes to generate a large number of insertion or deletion(indel) products. The large number of sequence outcomes that arise fromCas9 cutting allows for clear determination of sequences that have beencut by the nuclease. The ability to distinguish cut versus non-cutsequences has been used in two predominant ways.

First, the expression of the Cas9 nuclease and/or its single guide RNA(sgRNA) have been connected to cellular signals. Additionally, whetherthat signal has occurred based on sequence modifications to the Cas9targeted genomic locus has been recorded. However, this approach islimited because each signal requires a unique target locus, which makestracking the relative timing of multiple signals difficult to interpret.Another limitation to this approach is that multiple target loci aredesired for a specific sgRNA because generation of an indel oftenseverely hinders additional mutagenesis of the target locus; this oftenmeans that pre-engineered target loci are integrated into cells forediting instead of direct mutagenesis of endogenous loci.

Second, Cas9 indels have been used to track cellular lineages. Asdescribed herein, the large number of possible indel states generated byCas9 nuclease activity allows for the generation of cellulardevelopmental trees that suggest which cells have arisen from oneanother across time. This approach is a powerful way to understand howcells arise from one another and has been used to help identify uniquecell states and types across developmental time by performing RNAsequencing on selected cellular pools. This approach cannotindependently report on cellular signaling events, their order, and mayhave biases when reporting on pre-cursor versus terminallydifferentiated cell states.

Cas9 nuclease mediated lineage tracing and signal recording are powerfultechniques that come with some important caveats. Signal recording withCas9 nuclease is often very technically challenging. Cas9 cuttingexhausts the target locus (repeated cutting is difficult once an indelis generated) making it difficult to record long term stimuli at thesingle cell level. The kinetics of Cas9 cutting can be tuned to enablelonger term recording events though the ability to integrate the order,intensity, and duration of multiple stimuli remains a very challengingtechnical problem that may not be achievable with this tool. Cas9lineage tracing experiments have been incredibly powerful but sufferfrom minor technical challenges of sequence collapse due to simultaneousCas9 cuts at a target locus. These lineage-tracing experiments requireediting of pre-designed target loci, limiting the flexibility of thisapproach.

DNA base editing has also been used to track cellular signaling events.Base editing is not well suited for lineage tracing due to the lownumber of outcome states generated by an editing event relative to thenumber of states generated by Cas9 indels; however, the pre-definednature of the sequence modifications made by a base editor areparticularly useful for tracking internal and external cellular stimuli.As described herein, either base editor or sgRNA expression can beconnected to a particular biological or chemical stimulus. Base editingactivity has been used to track a large number of individual stimuli inboth mammalian and bacterial cells. This approach has also been used totrack consecutive stimuli where a first editing event was necessarybefore a second edit could take place.

Base editing signal recording is an important first step for the field,but it has a number of limitations. One such limitation is that baseediting exhausts its target after editing, limiting the dynamic range ofthe technique. This means that using endogenous targets for recordingevents is often difficult and limited to recording bulk activitiesinstead of activity at the single cell level. An alternative to this isthe introduction of a pre-designed repetitive recording locus thoughthis has not been performed to date. There are also issues withtwo-signal recording. These two signal-recording experiments only reporton the presence of the second stimulus after a first; it does not reportwhich stimulus happened first or how long the stimuli were present. Thisfundamentally limits the biological understanding gleaned from theexperiment.

It has been proposed that PE lineage tracing can do both lineage tracingand cellular signaling recording by modifying genomic target sequencesas well as integrated pre-designed sequences. PE uses a synthetic fusionprotein comprising a Cas9 nickase fragment (often the SpCas9 H840Avariant) and a reverse transcriptase (RT) domain, along with anengineered prime editing guide RNA (PEgRNA). Together, these componentstarget a specific genomic sequence and install a pre-determined edit.Since the PEgRNA specifies both the target genomic sequence and theediting outcome, highly specific and controlled genome modification canbe achieved simultaneously using multiple PEgRNAs within the same cell.Accessible genome modifications include all single nucleotidesubstitution, small to medium size sequence insertions, and small tomedium size sequence deletions. The versatility of this genome editingtechnology should enable temporally coupled, signal-specific recordingwithin cells.

Utility of PE Lineage and Cell Signaling Recording

Recording cellular signaling can be accomplished in a number of ways.One important first application of this approach is to connect DNAmodification events to cell cycle associated signals like the expressionof cyclins, CDKs, or other proteins specific to phases of the cellularlife span one could generate a cellular clock. A cellular clock allowsresearchers to understand the order of various signals being receivedand processed by individual cells. A molecular clock would also enablethe determination of long-term signaling versus short-term bursts ofsignaling. Using prime editing components that can only edit once a cellcycle could also lead to a molecular clock. If editing can only proceedonce a cell cycle without continued DNA modification (perhaps by notnicking the non-edited DNA strand) one could imagine a system that onlycan be edited by a second targeting PEsgRNA in a subsequent celldivision. PE is particularly useful as a cellular clock as it canrepeatedly insert, delete, or mutate loci in a predetermined way withinsertions being particularly valuable as repeated, regular insertionscan be made at any target genomic locus.

Another important application related to recording cellular signals isthe parallel recording of a large number of cellular inputs. Linkingcellular signaling events to DNA modification enables recording ofwhether such a signaling event has occurred. Similar to Cas9nuclease-based or base editing-based recording systems, recording ofcellular events can be tethered to gRNA or editor expression. Unlikethese other approaches, lineage prime editing should be able to recordthe order, intensity, and duration of signaling events without requiringstrict sequence motifs for ordered editing. Indeed, lineage primeediting should be able to integrate the cellular counter described abovewith signal specific insertions, deletions, or mutations to study theorder, intensity, and duration of biological signals. Due to theprogrammable nature of prime editing, this approach can be achieved atgenomic loci pre-existing in the target cell of interest (whether thisis in bacteria, mice, rats, monkeys, pigs, humans, zebrafish, C.elegans, etc.). It is important to note as well that prime editingrecording installs barcodes in a guide RNA dependent manner, the numberof inputs is limited to the number of signals for which there arereliable signal specific guide RNA expression cassettes (which should bevery high due to the ability to tether these expressions to the activityof RNA Pol II promoters). The number of recordable signals scaleslinearly with the number of PEgRNAs needed.

PE can also be used to trace cellular lineages. Repeated sequencemodification can be used to generate unique cellular barcodes to trackindividual cells. The arrays of barcodes, their order, and size can allbe used to infer cellular lineages in a way that can be complementary tothe large number of indel states generated by Cas9 nuclease.

Prime Editing Methodology for Repeated Sequence Modification

A number of distinct modalities for repeated sequence modification usingprime editing (PE) were envisioned: DNA mutagenesis; sequence deletion;and, sequence insertion. Notably, these applications can be used oneither pre-existing genomic DNA targets or on pre-designed DNA sequencesthat researchers integrate into target cells. These techniques of serialsequence modification have value for recording information and fordesigned or stochastic modification of target loci in a continuousmanner. Serial targeted locus modification may be particularly usefulfor generating libraries of variants in various hosts.

Repeated sequence mutation can be used to alter either genomic DNA orpre-designed integrated DNA sequences in an iterative manner to reporton cellular signaling events. In this paradigm, mutations installed byPE gRNA activity will correspond to the presence of a cellular signal.These point mutations could install PAM motifs necessary for serialediting events, as well as point mutations that correspond to thepresence of specific signals. This system would require gRNA designprior to use as each successive guide RNA will use a novel protospacer;however, it could be especially powerful for examining individual orsmall numbers of stimuli of particular interest. Installation of thesemutations could be dependent on individual biological stimuli or couldbe connected to consistent cellular processes that would mark cellulartime. The sequences below correspond to SEQ ID NOs: 743, 744, 744, and745.

Target genomic DNA sequence CGTATCGGTAACTGATCCGATGGAAAGCCAGTTCAGAACCGTarget sequence post edit #1 (install G and T)CGTATCGGTAACTGATCCGATGGAATGCCAGGTCAGAACCGTarget sequence post edit #1 (use AGG PAM)CGTATCGGTAACTGATCCGATGGAATGCCAGGTCAGAACCGTarget sequence post edit #2 (install G and A)CGTATCGGTAACTGATCCGATGGAATGCCAGGTCAAAACGG

Another similar PE guide RNA-intensive method is the repeated deletionof target sequences. Removal of individual sequences from a target locuswould allow for the ability to reconstruct signaling events through theloss of DNA motifs. Design of PEgRNAs that delete successive sequenceswould enable the tracking of consecutive signals. This would allowresearchers to identify instances where one signal followed another,which would allow researchers to probe which signaling events happen inwhich order. Such a system has been tested using CAMERA; however, thisrequired pre-selection of particular loci with unique sequencerequirements. Successive sequence deletion using PE would allow for theparalleled recording of pairwise events in individual cells as nospecific sequence determinants are required. This would allowresearchers the ability to probe pairwise signaling events in amultiplexed manner inside any target cell of interest. The sequencesbelow correspond to SEQ ID NOs: 746, 747, and 748.

Example target DNA sequence CGTATCGGTAACTGATCCGATGGAAAGCCAGTTCAGAACCGTarget sequence post edit #1 (delete AAA)CGTATCGGTAACTGATCCGATGGGCCAGTTCAGAACCGTarget sequence post edit #2 (delete GCC)CGTATCGGTAACTGATCCGATGGAGTTCAGAACCG

Sequence insertion is a third approach for the tracking cellularsignaling events. Some variants of this strategy are lessPEgRNA-dependent than mutagenesis or deletion. A number of differentinsertion strategies exist-insertion of short sequences, insertion ofprotospacers, insertion of a protospacer and a barcode, insertion ofnovel homology sequences, and insertion of homology sequences with abarcode.

Insertion of short repetitive sequences is a way to incrementallyincrease the size of a target sequence to measure the passage of time ina cell. In this system insertion of 5 or more nucleotides of repetitivesequence can cause repeat expansion in connection to either the passageof time or the continued presence of a pre-determined stimulus. Thelocus-agnostic nature of lineage PE again enables paralleled tracking ofmultiple unique sequence expansions in connection with discretebiological signals. This should enable measuring of the intensity ofmultiple biological signals across cellular time in individual cells.The sequences below correspond to SEQ ID NOs: 749, 750, and 751.

Example target DNA sequence genomic sequence-CGTATCGTATCGTATCGTATTGG-genomic sequenceTarget sequence post edit #1 (install CGTAT) genomic sequence-CGTATCGTATCGTATCGTATCGTATTGG-genomic sequenceTarget sequence post edit #2 (install CGTAT) genomic sequence-CGTATCGTATCGTATCGTATCGTATCGTATTGG-genomic sequence

Insertion of different short sequences would require a handful ofPEgRNAs in a PGP-1%,DNA manner similar to the deletional space. Thenumber of signals being recorded and the size of the inserted sequencewould determine the number of combinations of PEgRNAs needed. Onechallenge with recording multiple sequences in this system would be thedifferent efficiencies of each PEgRNA at inserting its cargo sequence.

Insertion of protospacers as indicators of cellular signals is enticingthough technical challenges may infringe on the efficiency of thisapproach. Single PEgRNA systems will be challenging to use becausePEgRNA cassettes would be substrates for themselves, causing insertionsinto the PEgRNA that compromise the efficiency and fidelity ofsuccessive edits. This same problem persists for two or three PEgRNAsystems, as each guide is a substrate for another, enabling insertion ofother sequences into the guide cassette itself, which could lead toinappropriate insertion of protospacer sequences in connection with thewrong signal. These protospacer insertion systems are also difficult toimagine with the inclusion of barcode sequences. Single PEgRNA barcodesystems would simply write over the barcodes used, removing the datastored in the first edit. Multiple guide systems again suffer frominsertions into other PEgRNA expression constructs, limiting its utility(especially in vivo). See FIG. 90 .

Insertion of homology sequences (i.e., sequences 3′ of the Cas9 nicklocation), and especially homology sequences with associated barcodes,appear to be particularly useful lineage PE strategies. This systemavoids the issues associated with protospacer insertion by ensuring thatsuccessive rounds of editing result in the insertion of a barcode from aPEgRNA cassette that cannot be modified by other PEgRNA editing eventsin the same cell. The barcoding system is valuable as multiple barcodescan be associated with a given stimulus. This system preserves themajority of the target protospacer but alters the seed sequence, PAM,and downstream adjacent nucleotides. This enables multiple signals to beconnected to one editing locus without significant re-designing of thePEgRNAs being used. This strategy would enable multiplexed barcodeinsertion in response to a large number of cellular stimuli (eitherinternal or external) at a single locus. It could enable recording ofintensity, duration, and order of as many signals as there exist uniquebarcodes (which can be designed with multiple N nucleotides to generate4{circumflex over ( )}N possible barcodes; i.e. a 5-nt barcode wouldenable recording of 4{circumflex over ( )}5 or 1024 unique signals atonce). This system could be used both in vitro and in vivo.

REFERENCES CITED IN EXAMPLE 13

Each of the following references are incorporated herein by reference.

-   1. Recording development with single cell dynamic lineage tracing.    Aaron McKenna, James A. Gagnon.-   2. Whole-organism lineage tracing by combinatorial and cumulative    genome editing. Mckenna et al. Science. 2016 Jul. 29;    353(6298):aaf7907. doi10.1126/science.aaf7907. Epub 2016 May 26.-   3. Molecular recording of mammalian embryogenesis. Chan et al. 2019.    Nature. June; 570(7759):77-82. doi: 10.1038/s41586-019-1184-5. Epub    2019 May 13.

Example 14—Modulating Biomolecule Activity and/or Localization by PE

The subcellular localization and modification states of biomoleculesregulate their activities. Specific biological functions liketranscriptional control, cellular metabolism, and signal transductioncascades are all carefully orchestrated in particular locations withinthe cell. As such, modulating the cellular localization and modificationstates of proteins represents a potential therapeutic strategy for thetreatment of disease. Some existing therapeutics have been developed toalter the localization of target proteins. For example, farnesylationinhibitors are designed to prevent lipidation and membrane targeting ofimportant oncogenic proteins like KRAS. Similarly,small-molecule-induced ubiquitination of target proteins directs them tothe proteasome for degradation. The ability to traffic proteins to theseand other unique cellular compartments provides an opportunity to altera number of biological processes. It is proposed herein that the use ofprime editing (PE) to install genetically encoded handles for alteringthe modification state and the subcellular trafficking of biomoleculeswith a genetically encoded signal (e.g. proteins, lipids, sugars, andnucleic acids) for the purposes of therapeutics.

PE is a genome editing technology that enables the installation,deletion, or replacement of short DNA sequences into any genomic locustargetable with a Cas9 enzyme. Using this technology, one could inprinciple install or remove important DNA, RNA, or protein codingsequences that change the activities of these important biomolecules.More specifically, prime editing could be used to install motifs, orsignals, that change the localization or modification properties ofbiomolecules. Some examples include modification to: protein amino acidsequences; motifs for post translational modifications; RNA motifs thatchange folding or localization; and, installation of DNA sequences thatchange the local chromatin state or architecture of the surrounding DNA.

One target biomolecule for PE-mediated modification is DNA.Modifications to DNA could be made to install a number of DNA sequencesthat change the accessibility of the target locus. Chromatinaccessibility controls gene transcriptional output. Installation ofmarks to recruit chromatin compacting enzymes should decrease thetranscriptional output of neighboring genes, while installation ofsequences associated with chromatin opening should make regions moreaccessible and in turn increase transcription. Installation of morecomplex sequence motifs that mirror native regulatory sequences shouldprovide more nuanced and biologically sensitive control than thecurrently available dCas9 fusions to different epigenetic reader,writer, or eraser enzymes-tools that typically install large numbers ofa single type of mark that may not have a particular biologicalantecedent. Installation of sequences that will bring two loci intoclose proximity, or bring loci into contact with the nuclear membrane,should also alter the transcriptional output of those loci as has beendemonstrated in the burgeoning field of 3-D genomic architecture.

Modifications to RNAs can also be made to alter their activity bychanging their cellular localization, interacting partners, structuraldynamics, or thermodynamics of folding. Installation of motifs that willcause translational pausing or frameshifting could change the abundanceof mRNA species through various mRNA processing mechanisms. Modifyingconsensus splice sequences would also alter the abundance and prevalenceof different RNA species. Changing the relative ratio of differentsplice isoforms would predictably lead to a change in the ratio ofprotein translation products, and this could be used to alter manybiological pathways. For instance, shifting the balance of mitochondrialversus nuclear DNA repair proteins would alter the resilience ofdifferent cancers to chemotherapeutic reagents. Furthermore, RNAs couldbe modified with sequences that enable binding to novel protein targets.A number of RNA aptamers have been developed that bind with highaffinity to cellular proteins. Installation of one of these aptamerscould be used to either sequester different RNA species through bindingto a protein target that will prevent their translation, biologicalactivity, or to bring RNA species to specific subcellular compartments.Biomolecule degradation is another class of localization modification.For example, RNA methylation is used to regulate RNAs within the cell.Consensus motifs for methylation could be introduced into target RNAcoding sequences with PE. RNAs could also be modified to includesequences that direct nonsense mediated decay machinery or other nucleicacid metabolism pathways to degrade the target RNA species would changethe pool of RNAs in a cell. Additionally, RNA species could be modifiedto alter their aggregation state. Sequences could be installed on singleRNAs of interest or multiple RNAs to generate RNA tangles that wouldrender them ineffective substrates for translation or signaling.

Modifications to proteins via post-translational modification (PTM) alsorepresent an important class of biomolecule manipulation that can becarried out with PE. As with RNA species, changing the abundance ofproteins in a cell is an important capability of PE. Editing can be doneto install stop codons in an open reading frame—this will eliminatefull-length product from being produced by the edited DNA sequence.Alternatively, peptide motifs can be installed that cause the rate ofprotein degradation to be altered for a target protein. Installation ofdegradation tags into a gene body could be used to alter the abundanceof a protein in a cell. Moreover, introduction of degrons that areinduced by small molecules could enable temporal control over proteindegradation. This could have important implications for both researchand therapeutics as researchers could readily assess whether smallmolecule-mediated therapeutic protein degradation of a given target wasa viable therapeutic strategy. Protein motifs could also be installed tochange the subcellular localization of a protein. Amino acid motifs canbe installed to preferentially traffic proteins to a number ofsubcellular compartments including the nucleus, mitochondria, cellmembrane, peroxisome, lysosome, proteasome, exosome, and others.

Installing or destroying motifs modified by PTM machinery can alterprotein post-translational modifications. Phosphorylation,ubiquitylation, glycosylation, lipidation (e.g. farnesylation,myristoylation, palmitoylation, prenylation, GPI anchors),hydroxylation, methylation, acetylation, crotonylation, SUMOylation,disulfide bond formations, side chain bond cleavage events, polypeptidebackbone cleavage events (proteolysis), and a number of other proteinPTMs have been identified. These PTMs change protein function, often bychanging subcellular localization. Indeed, kinases often activatedownstream signaling cascades via phosphorylation events. Removal of thetarget phosphosite would prevent signal transduction. The ability tosite-specifically ablate or install any PTM motif while retainingfull-length protein expression would be an important advance for bothbasic research and therapeutics. The sequence installation scope andtarget window of PE make it well suited for broad PTM modificationspace.

Removal of lipidation sites should prevent the trafficking of proteinsto cell membranes. A major limitation to current therapeutics thattarget post-translational modification processes is their specificity.Farnesyl transferase inhibitors have been tested extensively for theirability to eliminate KRAS localization at cell membranes. Unfortunately,global inhibition of farnesylation comes with numerous off targeteffects that have prevented broad use of these small molecules.Similarly, specific inhibition of protein kinases with small moleculescan be very challenging due to the large size of the human genome andsimilarities between various kinases. PE offers a potential solution tothis specificity problem, as it enables inhibition of modification ofthe target protein by ablation of the modification site instead ofglobal enzyme inhibition. For example, removal of the lapidated peptidemotif in KRAS would be a targeted approach that could be used in placeof farnesyl transferase inhibition. This approach is the functionalinverse of inhibiting a target protein activity by installing alipid-targeting motif on a protein not designed to be membrane bound.

PE can also be used to instigate protein-protein complexation events.Proteins often function within complexes to execute their biologicalactivity. PE can be used to either create or destroy the ability ofproteins to exist within these complexes. To eliminate complex formationevents, amino acid substitutions or insertions along the protein:protein interface could be installed to disfavor complexation. SSX18 isa protein component of the BAF complex, an important histone-remodelingcomplex. Mutations in SSX18 drive synovial sarcomas. PE could be used toinstall side chains that prevent SSX18 from binding to its proteinpartners in the complex to prevent its oncogenic activity. PE could alsobe used to remove the pathogenic mutations to restore WT activity ofthis protein. Alternatively, PE could be used to keep proteins withineither their native complex or to drag them to participate ininteractions that are unrelated to their native activity to inhibittheir activity. Forming complexes that maintain one interaction stateover another could represent an important therapeutic modality. Alteringprotein: protein interfaces to decrease the Kd of the interaction wouldkeep those proteins stuck to one another longer. As protein complexescan have multiple signaling complexes, like n-myc driving neuroblastomasignaling cascades in disease but otherwise participating in healthytranscriptional control in other cells. PE could be used to installmutations that drive n-myc association with healthy interactionspartners and decrease its affinity for oncogenic interaction partners.

REFERENCES CITED IN EXAMPLE 14

Each of the following references are incorporated herein by reference.

-   1. Selective Target Protein Degradation via Phthalimide Conjugation.    Winter et al. Science. Author manuscript; available in PMC 2016 Jul.    8.-   2. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX    oncogenic fusion in synovial sarcoma. Kadoch and Crabtree. Cell.    2013 Mar. 28; 153(1):71-85. doi: 10.1016/j.cell.2013.02.036.-   3. Ribosomal frameshifting and transcriptional slippage: From    genetic steganography and cryptography to adventitious use. Atkins    et al. Nucleic Acids Research, Volume 44, Issue 15, 6 Sep. 2016,    Pages 7007-7078.-   4. Transcriptional Regulation and its Misregulation in Disease. Lee    and Young. Cell. Author manuscript; available in PMC 2014 Mar. 14.-   5. Protein localization in disease and therapy. Mien-Chie Hung,    Wolfgang Link Journal of Cell Science 2011 124: 3381-3392.-   6. Loss of post-translational modification sites in disease. Li et    al. Pac Symp Biocomput. 2010:337-47. PTMD: A Database of Human    Disease-associated Post-translational Modifications. Xu et al.    Genomics Proteomics Bioinformatics. 2018 August; 16(4):244-251. Epub    2018 Sep. 21.-   7. Post-transcriptional gene regulation by mRNA modifications. Zhao    et al. Nature Reviews Molecular Cell Biology volume 18, pages 31-42    (2017).

Example 15—Design and Engineering of PEgRNAs

Described herein is a series of PEgRNA designs and strategies that canimprove prime editing (PE) efficiency.

Prime editing (PE) is a genome editing technology that can replace,insert, or remove defined DNA sequences within a targeted genetic locususing information encoded within a prime editing guide RNA (PEgRNA).Prime editors (PEs) consist of a sequence-programmable DNA bindingprotein with nuclease activity (Cas9) fused to a reverse transcriptase(RT) enzyme. PEs form complexes with PEgRNAs, which contain theinformation for targeting specific DNA loci within their spacersequences, as well as information specifying the desired edit in anengineered extension built into a standard sgRNA scaffold. PE:PEgRNAcomplexes bind and nick the programmed target DNA locus, allowinghybridization of the nicked DNA strand to the engineered primer bindingsequence (PBS) of the PEgRNA. The reverse transcriptase domain thencopies the edit-encoding information within the RT template portion ofthe PEgRNA, using the nicked genomic DNA as a primer for DNApolymerization. Subsequent DNA repair processes incorporate the newlysynthesized edited DNA strand into the genomic locus. While theversatility of prime editing holds great promise as a research tool andpotential therapeutic, several limitations in efficiency and scope existdue to the multi-step process required for editing. For example,unfavorable RNA structures that form within the PEgRNA can inhibit thecopying of DNA edits from the PEgRNA to the genomic locus. One potentialway to improve PE technology is through redesign and engineering of thecritical PEgRNA component. Improvements to the design of these PEgRNAsare likely to be necessary for improved PE efficiency, as well as enableinstallation of longer inserted sequences into the genome.

Described herein is a series of PEgRNA designs that are envisioned toimprove the efficacy of PE. These designs take advantage of a number ofpreviously published approaches for improving sgRNA efficacy and/orstability, as well as utilize a number of novel strategies. Theseimprovements can belong to one or more of a number of differentcategories:

-   -   (1) Longer PEgRNAs. This category relations to improved designs        that enable efficient expression of functional PEgRNAs from        non-polymerase III (pol III) promoters, which would enable the        expression of longer PEgRNAs without burdensome sequence        requirements;    -   (2) Core Improvements. This category relates to improvements to        the core, Cas9-binding PEgRNA scaffold, which could improve        efficacy;    -   (3) RT processivity. This category relates to modifications to        the PEgRNA that improve RT processivity, enabling the insertion        of longer sequences at targeted genomic loci; and    -   (4) Termini motifs. This category relates to the addition of RNA        motifs to the 5′ and/or 3′ termini of the PEgRNA that improve        PEgRNA stability, enhance RT processivity, prevent mis-folding        of the PEgRNA, or recruit additional factors important for        genome editing.

Described herein are a number of potential such PEgRNA designs in eachcategory. Several of these designs have been previously described forimproving sgRNA activity with Cas9 and are indicated as such. Describedherein is also a platform for the evolution of PEgRNAs for givensequence targets that would enable the polishing of the PEgRNA scaffoldand enhance PE activity (5). Notably, these designs could also bereadily applied to improve PEgRNAs recognized by any Cas9 or evolvedvariant thereof.

(1) Longer PEgRNAs.

sgRNAs are typically expressed from the U6 snRNA promoter. This promoterrecruits pol II to express the associated RNA and is useful forexpression of short RNAs that are retained within the nucleus. However,pol III is not highly processive and is unable to express RNAs longerthan a few hundred nucleotides in length at the levels required forefficient genome editing¹⁸³. Additionally, pol III can stall orterminate at stretches of U's, potentially limiting the sequencediversity that could be inserted using a PEgRNA. Other promoters thatrecruit polymerase II (such as pCMV) or polymerase I (such as the U1snRNA promoter) have been examined for their ability to express longersgRNAs¹⁸³. However, these promoters are typically partially transcribed,which would result in extra sequence 5′ of the spacer in the expressedPEgRNA, which has been shown to result in markedly reduced Cas9:sgRNAactivity in a site-dependent manner. Additionally, while polII-transcribed PEgRNAs can simply terminate in a run of 6-7 U's, PEgRNAstranscribed from pol II or pol I would require a different terminationsignal. Often such signals also result in polyadenylation, which wouldresult in undesired transport of the PEgRNA from the nucleus. Similarly,RNAs expressed from pol II promoters such as pCMV are typically5′-capped, also resulting in their nuclear export.

Previously, Rinn and coworkers screened a variety of expressionplatforms for the production of long-noncoding RNA-(lncRNA) taggedsgRNAs¹⁸³. These platforms include RNAs expressed from pCMV and thatterminate in the ENE element from the MALAT1 ncRNA from humans¹⁸⁴, thePAN ENE element from KSHV¹⁸⁵, or the 3′ box from U1 snRNA¹⁸⁶. Notably,the MALAT1 ncRNA and PAN ENEs form triple helices protecting thepolyA-tail^(184, 187). It is anticipated that, in addition to enablingexpression of RNAs, these constructs could also enhance RNA stability(see section iv). Using the promoter from the U1 snRNA to enableexpression of these longer sgRNAs¹⁸³ was also explored. It isanticipated that these expression systems will also enable theexpression of longer PEgRNAs. In addition, a series of methods have beendesigned for the cleavage of the portion of the pol II promoter thatwould be transcribed as part of the PEgRNA, adding either aself-cleaving ribozyme such as the hammerhead¹⁸⁸, pistol¹⁸⁹, hatchet¹⁸⁹,hairpin¹⁹⁰, VS¹⁹¹, twister¹⁹², or twister sister¹⁹² ribozymes, or otherself-cleaving elements to process the transcribed guide, or a hairpinthat is recognized by Csy4¹⁹³ and also leads to processing of the guide.Also, it is hypothesized that incorporation of multiple ENE motifs couldlead to improved PEgRNA expression and stability, as previouslydemonstrated for the KSHV PAN RNA and element¹⁸⁵. It is also anticipatedthat circularizing the PEgRNA in the form of a circular intronic RNA(ciRNA) could also lead to enhanced RNA expression and stability, aswell as nuclear localization.¹⁹⁴

PEgRNA expression platform consisting of pCMV, Csy4hairping, the PEgRNA, and MALAT1 ENE (SEQ ID NO: 757)TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGATGCTGGTGGTTGGCACTCCTGGTTTCCAGGACGGGGTTCAAATCCCTGCGGCGTCTTTGCTTTGACTPEgRNA expression platform consisting of pCMV, Csy4hairping, the PEgRNA, and PAN ENE (SEQ ID NO: 758)TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAAPEgRNA expression platform consisting of pCMV, Csy4hairping, the PEgRNA, and 3xPAN ENE (SEQ ID NO: 759)TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAAACACACTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAATCTCTCTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAAPEgRNA expression platform consisting of pCMV, Csy4hairping, the PEgRNA, and 3′ box (SEQ ID NO: 760)TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAAPEgRNA expression platform consisting of pU1, Csy4hairping, the PEgRNA, and 3′ box (SEQ ID NO: 761)CTAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGGAAAAAGGGAGAGGCAGACGTCACTTCCCCTTGGCGGCTCTGGCAGCAGATTGGTCGGTTGAGTGGCAGAAAGGCAGACGGGGACTGGGCAAGGCACTGTCGGTGACATCACGGACAGGGCGACTTCTATGTAGATGAGGCAGCGCAGAGGCTGCTGCTTCGCCACTTGCTGCTTCACCACGAAGGAGTTCCCGTGCCCTGGGAGCGGGTTCAGGACCGCTGATCGGAAGTGAGAATCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCGGGAGTGCGCGGGGCAAGTGACCGTGTGTGTAAAGAGTGAGGCGTATGAGGCTGTGTCGGGGCAGAGGCCCAAGATCTCAGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTCAGCAAGTTCAGAGAAATCTGAACTTGCTGGATTTTTGGAGCAGGGAGATGGAATAGGAGCTTGCTCCGTCCACTCCACGCATCGACCTGGTATTGCAGTACCTCCAGGAACGGTGCACCCACTTTCTGGAGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAA

(2) Core Improvements.

The core, Cas9-binding PEgRNA scaffold can likely be improved to enhancePE activity. Several such approaches have already been demonstrated. Forinstance, the first pairing element of the scaffold (P1) contains aGTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such runs of Ts have beenshown to result in pol III pausing and premature termination of the RNAtranscript. Rational mutation of one of the T-A pairs to a G-C pair inthis portion of P1 has been shown to enhance sgRNA activity, suggestingthis approach would also be feasible for PEgRNAs¹⁹⁵. Additionally,increasing the length of P1 has also been shown to enhance sgRNA foldingand lead to improved activity¹⁹⁵, suggesting it as another avenue forthe improvement of PEgRNA activity. Finally, it is likely the polishingof the PEgRNA scaffold through directed evolution of PEgRNAs on a givenDNA target would also result in improved activity. This is described insection (v).

PEgRNA containing a 6 nt extension to P1 (SEQ ID NO: 228)GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTTPEgRNA containing a T-A to G-C mutation within P1 (SEQ ID NO: 229)GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT

(iii) Improvement of RT Processivity Via Modifications to the TemplateRegion of the PEgRNA

As the size of the insertion templated by the PEgRNA increases, it ismore likely to be degraded by endonucleases, undergo spontaneoushydrolysis, or fold into secondary structures unable to bereverse-transcribed by the RT or that disrupt folding of the PEgRNAscaffold and subsequent Cas9-RT binding. Accordingly, it is likely thatmodification to the template of the PEgRNA might be necessary to affectlarge insertions, such as the insertion of whole genes. Some strategiesto do so include the incorporation of modified nucleotides within asynthetic or semi-synthetic PEgRNA that render the RNA more resistant todegradation or hydrolysis or less likely to adopt inhibitory secondarystructures¹⁹⁶. Such modifications could include 8-aza-7-deazaguanosine,which would reduce RNA secondary structure in G-rich sequences;locked-nucleic acids (LNA) that reduce degradation and enhance certainkinds of RNA secondary structure; 2′-O-methyl, 2′-fluoro, or2′-O-methoxyethoxy modifications that enhance RNA stability. Suchmodifications could also be included elsewhere in the PEgRNA to enhancestability and activity. Alternatively or additionally, the template ofthe PEgRNA could be designed such that it both encodes for a desiredprotein product and is also more likely to adopt simple secondarystructures that are able to be unfolded by the RT. Such simplestructures would act as a thermodynamic sink, making it less likely thatmore complicated structures that would prevent reverse transcriptionwould occur. Finally, one could also imagine splitting the template intotwo, separate PEgRNAs. In such a design, a PE would be used to initiatetranscription and also recruit a separate template RNA to the targetedsite via an RNA-binding protein fused to Cas9 or an RNA recognitionelement on the PEgRNA itself such as the MS2 aptamer. The RT couldeither directly bind to this separate template RNA, or initiate reversetranscription on the original PEgRNA before swapping to the secondtemplate. Such an approach could enable long insertions by bothpreventing mis-folding of the PEgRNA upon addition of the long templateand also by not requiring dissociation of Cas9 from the genome for longinsertions to occur, which could possibly be inhibiting PE-based longinsertions.

(iv) Installation of Additional RNA Motifs at the 5′ or 3′ Termini

PEgRNA designs could also be improved via the installation of additionalmotifs at either end of the terminus of the RNA. Several suchmotifs—such as the PAN ENE from KSHV and the ENE from MALAT1 werediscussed earlier in part (i)^(184,185) as possible means to terminateexpression of longer PEgRNAs from non-pol III promoters. These elementsform RNA triple helices that engulf the polyA tail, resulting in theirbeing retained within the nucleus^(184,187). However, by forming complexstructures at the 3′ terminus of the PEgRNA that occlude the terminalnucleotide, these structures would also likely help preventexonuclease-mediated degradation of PEgRNAs. Other structural elementsinserted at the 3′ terminus could also enhance RNA stability, albeitwithout enabling termination from non-pol III promoters. Such motifscould include hairpins or RNA quadruplexes that would occlude the 3′terminus¹⁹⁷, or self-cleaving ribozymes such as HDV that would result inthe formation of a 2′-3′-cyclic phosphate at the 3′ terminus and alsopotentially render the PEgRNA less likely to be degraded byexonucleases¹⁹⁸. Inducing the PEgRNA to cyclize via incompletesplicing—to form a ciRNA—could also increase PEgRNA stability and resultin the PEgRNA being retained within the nucleus¹⁹⁴.

Additional RNA motifs could also improve RT processivity or enhancePEgRNA activity by enhancing RT binding to the DNA-RNA duplex. Additionof the native sequence bound by the RT in its cognate retroviral genomecould enhance RT activity¹⁹⁹. This could include the native primerbinding site (PBS), polypurine tract (PPT), or kissing loops involved inretroviral genome dimerization and initiation of transcription¹⁹⁹.Addition of dimerization motifs—such as kissing loops or a GNRAtetraloop/tetraloop receptor pair²⁰⁰—at the 5′ and 3′ termini of thePEgRNA could also result in effective circularization of the PEgRNA,improving stability. Additionally, it is envisioned that addition ofthese motifs could enable the physical separation of the PEgRNA spacerand primer, prevention occlusion of the spacer which would hinder PEactivity. Short 5′ extensions to the PEgRNA that form a small toeholdhairpin in the spacer region could also compete favorably against theannealing region of the PEgRNA binding the spacer. Finally, kissingloops could also be used to recruit other template RNAs to the genomicsite and enable swapping of RT activity from one RNA to the other(section iii).

PEgRNA-HDV fusion (SEQ ID NO: 230)GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT PEgRNA-MMLV kissing loop(SEQ ID NO: 231) GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGA GACGTCCCACCTTTTTTTPEgRNA-VS ribozyme kissing loop (SEQ ID NO: 232)GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTCCATCAGTTGACACCCTGAGGTTTTTTT PEgRNA-GNRA tetraloop/tetraloop receptor(SEQ ID NO: 233) GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTUACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTTPEgRNA template switching secondary RNA-HDV fusion (SEQ ID NO: 234)TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT

(v) Evolution of PEgRNAs

It is likely that the PEgRNA scaffold can be further improved viadirected evolution, in an analogous fashion to how SpCas9 and baseeditors have been improved²⁰¹. Directed evolution could enhance PEgRNArecognition by Cas9 or evolved Cas9 variants. Additionally, it is likelythat different PEgRNA scaffold sequences would be optimal at differentgenomic loci, either enhancing PE activity at the site in question,reducing off-target activities, or both. Finally, evolution of PEgRNAscaffolds to which other RNA motifs have been added would almostcertainly improve the activity of the fused PEgRNA relative to theunevolved, fusion RNA. For instance, evolution of allosteric ribozymescomposed of c-di-GMP-I aptamers and hammerhead ribozymes led todramatically improved activity²⁰², suggesting that evolution wouldimprove the activity of hammerhead-PEgRNA fusions as well. In addition,while Cas9 currently does not generally tolerate 5′ extension of thesgRNA, directed evolution will likely generate enabling mutations thatmitigate this intolerance, allowing additional RNA motifs to beutilized.

As described herein, a number of these approaches have already beendescribed for use with Cas9:sgRNA complexes, but no designs forimproving PEgRNA activity have been reported. Other strategies for theinstallation of programmable mutations into the genome includebase-editing, homology-directed recombination (HDR), precisemicrohomology-mediated end-joining (MMEJ), or transposase-mediatedediting. However, all of these approaches have significant drawbackswhen compared to PEs. Current base editors, while more efficient thanexisting PEs, can only install certain classes of genomic mutations andcan result in additional, undesired nucleotide conversions at the siteof interest. HDR is only feasible in a very small minority of cell typesand results in comparably high rates of random insertion and deletionmutations (indels). Precise MMEJ can lead to predictable repair ofdouble-strand breaks, but is largely limited to installation ofdeletions, is very site-dependent, and can also have comparably highrates of undesired indels. Transposase-mediated editing has to date onlybeen shown to function in bacteria. As such improvements to PE representpossibly the best path forward for the therapeutic correction of awide-swatch of genomic mutations.

REFERENCES CITED IN EXAMPLE 15

Each of the following references are cited in Example 15, each of whichare incorporated herein by reference.

-   1. Schechner, D M, Hacisuleyman E., Younger S T, Rinn J L. Nat    Methods 664-70 (2015).-   2. Brown J A, et al. Nat Struct Mol Biol 633-40 (2014).-   3. Conrad N A and Steitz J A. EMBO J 1831-41 (2005).-   4. Bartlett J S, et al. Proc Natl Acad Sci USA 8852-7 (1996).-   5. Mitton-Fry R M, DeGregorio S J, Wang J, Steitz T A, Steitz J A.    Science 1244-7 (2010).-   6. Forster A C, Symons R H. Cell. 1987.-   7. Weinberg Z, Kim P B, Chen T H, Li S, Harris K A, Lünse C E,    Breaker R R. Nat. Chem. Biol. 2015.-   8. Feldstein P A, Buzayan J M, Bruening G. Gene 1989.-   9. Saville B J, Collins R A. Cell. 1990.-   10. Roth A, Weinberg Z, Chen A G, Kim P G, Ames T D, Breaker R R.    Nat Chem Biol. 2013.-   11. Borchardt E K, et al. RNA 1921-30 (2015).-   12. Zhang Y, et al. Mol Cell 792-806 (2013).-   13. Dang Y, et al. Genome Biol 280 (2015).-   14. Schaefer M, Kapoor U, and Jantsch M F. Open Biol 170077 (2017).-   15. Nahar S, et al. Chem Comm 2377-80 (2018).-   16. Gao Y and Zhao Y. J Integr Plant Biol 343-9 (2014).-   17. Dubois N, Marquet R, Paillart J, Bernacchi S. Front Microbiol    527 (2018).-   18. Costa M and Michel F. EMBO J 1276-85 (1995).-   19. Hu J H, et al. Nature 57-63 (2018).-   20. Furukawa K, Gu H, Breaker R R. Methods Mol Biol 209-20 (2014).

Example 16—Expanding the Targeting Scope of PE Using DNA BindingProteins Other than SpCas9

Prime editing (PE) using Streptococcus pyogenes Cas9 (SpCas9) canefficiently install all single base substitutions, insertions,deletions, and combinations thereof at genomic loci where there is asuitably-placed NGG protospacer adjacent motif (PAM) that SpCas9 canefficiently bind. The methods described herein broaden the targetingcapability of PE by expanding the accessible PAMs and, therefore, thetargetable genomic loci accessible for efficient PE. Prime editors usingRNA-guided DNA binding proteins other than SpCas9 enable an expandedtargetable scope of genomic loci by allowing access to different PAMs.In addition, use of RNA-guided DNA binding proteins smaller than SpCas9also allows for more efficient viral delivery. PE with Cas proteins orother RNA-guided DNA binding proteins beyond SpCas9 will allow for highefficiency therapeutic edits that were either inaccessible orinefficient using SpCas9-based PE.

This is expected to be used in situations where SpCas9-based PE iseither inefficient due to non-ideal spacing of an edit to relative to anNGG PAM or the overall size of the SpCas9-based construct is prohibitivefor cellular expression and/or delivery. Specific disease-relevant locisuch as the Huntingtin gene, which has few and poorly located NGG PAMsfor SpCas9 near the target region, can easily be targeted usingdifferent Cas proteins in the PE system such as SpCas9-VRQR whichrecognizes an NGA PAM. Smaller Cas proteins will be used to generatesmaller PE constructs that can be packaged into AAV vectors moreefficiently, enabling better delivery to target tissues. FIG. 61 showsthe reduction to practice of prime editing using Staphylococcus aureusCRISPR-Cas as the RNA-guided DNA binding protein. NT is untreatedcontrol.

FIGS. 62A-62B provide a demonstration of the importance of theprotospacer for efficient installation of a desired edit at a preciselocation with prime editing. This highlights the importance of alternatePAMs and protospacers as novel features of this technology. “n.d.” inFIG. 62A is “not detected.”

FIG. 63 shows the reduction to practice of PE using SpCas9(H840A)-VRQRand SpCas9(H840A)-VRER as the RNA-guided DNA binding protein in a primeeditor system. The SpCas9(H840A)-VRQR napDNAbp is disclosed herein asSEQ ID NO: 87. The SpCas9(H840A)-VRER napDNAbp is disclosed herein asSEQ ID NO: 88. The SpCas9(H840A)-VRER-MMLV RT fusion protein isdisclosed herein as SEQ ID NO: 516, wherein the MMLV RT comprises theD200N, L603W, T330P, T306K, and W313F substitutions relative to the wildtype MMLV RT. The SpCas9(H840A)-VRQR-MMLV RT fusion protein is disclosedherein as SEQ ID NO: 515, wherein the MMLV RT comprises the D200N,L603W, T330P, T306K, and W313F substitutions relative to the wild typeMMLV RT. Seven different loci in the human genome are targeted: 4 withthe SpCas9(H840A)-VRQR-MMLV RT prime editor system and 3 with theSpCas9(H840A)-VRER-MMLV RT system. The amino acid sequences of thetested constructs are as follows:

SACAS9-M-MLV MKRTADGSEFESPKKKRKVGKRNYILGLDIGITSVGYGIIDYETRRT PRIME EDITOR DVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKR KV (SEQ ID NO: 660)SPCAS9(H840A)- MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY VRQR-MALONEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR MURINERRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH LEUKEMIA VIRUSERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL REVERSEAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN TRANSCRIPTASEASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL PRIME EDITORGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 661) SPCAS9(H840A)-MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY VRER-MALONEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR MURINERRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH LEUKEMIA VIRUSERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL REVERSEAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN TRANSCRIPTASEASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL PRIME EDITORGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 662)

As shown in FIG. 63 , the SpCas9(H840A)-VRQR-MMLV RT was operational atPAM sites that included “AGAG” and “GGAG”, with some editing activity at“GGAT” and “AGAT” PAM sequences. The SpCas9(H840A)-VRER-MMLV RT wasoperational at PAM sites that included “AGCG” and “GGCG”, with someediting activity at “TGCG.”

The data demonstrates that prime editing may be conducted usingnapDNAbps which bear different PAM specificities, such as those Cas9variant described herein.

Example 17—Introduction of Recombinase Target Sites with PE

This Example describes a method to address genetic disease or generatetailor-made animal or plant models by using prime editing (PE) tointroduce recombinase targets sites (SSR target sites) in mammalian andother genomes with high specificity and efficiency.

This Example describes use of PE to introduce recombinase recognitionsequences at high-value loci in human or other genomes, which, afterexposure to site-specific recombinase(s), will direct precise andefficient genomic modifications (FIG. 64 ). In various embodiments showin FIG. 64 , PE may be used to (b) insert a single SSR target for use asa site for genomic integration of a DNA donor template. (c) shows how atandem insertion of SSR target sites can be used to delete a portion ofthe genome. (d) shows how a tandem insertion of SSR target sites can beused to invert a portion of the genome. (e) shows how the insertion oftwo SSR target sites at two distal chromosomal regions can result inchromosomal translocation. (f) shows how the insertion of two differentSSR target sites in the genome can be used to exchange a cassette from aDNA donor template. Each of the types of genome modifications areenvisioned by using PE to insert SSR targets, but this list also is notmeant to be limiting.

Many large-scale genomic changes, such as gene insertions, deletions,inversions, or chromosomal translocations, are implicated in geneticdisease¹⁻⁷. In addition, custom and targeted manipulation of eukaryoticgenomes is important for research into human disease, as well asgeneration of transgenic plants^(8,9) or other biotechnologicalproducts. For example, microdeletions of chromosomes can lead todisease, and replacement of these deletions by insertions of criticalDNA elements could lead to a permanent amelioration of disease. Inaddition, diseases resulting from inversions, gene copy number changes,or chromosomal translocations could be addressed by restoring theprevious gene structure in affected cells. Alternatively, in plants orother high value eukaryotic organisms used in industry, introduction ofrecombinant DNA or targeted genomic rearrangements could lead toimproved products, for example crops which require fewer resources orare resistant to pathogens. Current technologies for effectinglarge-scale genomic changes rely on random or stochastic processes, forexample the use of transposons or retroviruses, while other desiredgenomic modifications have only been achieved by homologousrecombination strategies.

One appealing class of proteins for accomplishing targeted and efficientgenomic modification is site-specific recombinases (SSRs). SSRs have along history of being used as a tool for genomic modification¹⁰⁻¹³. SSRsare considered promising tools for gene therapy because they catalyzethe precise cleavage, strand exchange, and rejoining of DNA fragments atdefined recombination targets¹⁴ without relying on the endogenous repairof double-strand breaks which can induce indels, translocations, otherDNA rearrangements, or p53 activation¹⁵⁻¹⁸. The reactions catalyzed bySSRs can result in the direct replacement, insertion, or deletion oftarget DNA fragments with efficiencies exceeding those ofhomology-directed repair^(14,19).

Although SSRs offer many advantages, they are not widely used becausethey have a strong innate preference for their cognate target sequence.The recognition sequences of SSRs are typically ≥20 base pairs and thusunlikely to occur in the genomes of humans or model organisms. Further,the native substrate preferences of SSRs are not easily altered, evenwith extensive laboratory engineering or evolution²⁰. This limitation isovercome by using PE to directly introduce recombinase targets into thegenome, or to modify endogenous genomic sequences which nativelyresemble recombinase targets. Subsequent exposure of the cell torecombinase protein will permit precise and efficient genomicmodification directed by the location and orientation of the recombinasetarget(s) (FIG. 64 ).

PE-mediated introduction of recombinase targets could be particularlyuseful for the treatment of genetic diseases which are caused bylarge-scale genomic defects, such as gene loss, inversion, orduplication, or chromosomal translocation¹⁻⁷ (Table 6). For example,Williams-Beuren syndrome is a developmental disorder caused by adeletion of 24 in chromosome 721. No technology exists currently for theefficient and targeted insertion of multiple entire genes in livingcells (the potential of PE to do such a full-length gene insertion iscurrently being explored but has not yet been established); however,recombinase-mediated integration at a target inserted by PE offers oneapproach towards a permanent cure for this and other diseases. Inaddition, targeted introduction of recombinase recognition sequencescould be highly enabling for applications including generation oftransgenic plants, animal research models, bioproduction cell lines, orother custom eukaryotic cell lines. For example, recombinase-mediatedgenomic rearrangement in transgenic plants at PE-specific targets couldovercome one of the bottlenecks to generating agricultural crops withimproved properties^(8,9).

A number of SSR family members have been characterized and their targetsequences described, including natural and engineered tyrosinerecombinases (TABLE 7), large serine integrases (TABLE 8), serineresolvases (TABLE 9), and tyrosine integrases (TABLE 10). Modifiedtarget sequences that demonstrate enhanced rates of genomic integrationhave also been described for several SSRs²²⁻³⁰. In addition to naturalrecombinases, programmable recombinases with distinct specificities havebeen developed³¹⁻⁴⁰. Using PE, one or more of these recognitionsequences could be introduced into the genomic at a specified location,such as a safe harbor locus⁴¹⁻⁴³, depending on the desired application.

For example, introduction of a single recombinase target in the genomewould result in integrative recombination with a DNA donor template(FIG. 64B). Serine integrases, which operate robustly in human cells,may be especially well-suited for gene integration^(44,45).Additionally, introduction of two recombinase targets could result indeletion of the intervening sequence, inversion of the interveningsequence, chromosomal translocation, or cassette exchange, depending onthe identity and orientation of the targets (FIGS. 64C-64F). By choosingendogenous sequences that already closely resemble recombinase targets,the scope of editing required to introduce the complete recombinasetarget would be reduced.

Finally, several recombinases have been demonstrated to integrate intohuman or eukaryotic genomes at natively occurring pseudosites⁴⁶⁻⁶⁴. PEediting could be used to modify these loci to enhance rates ofintegration at these natural pseudosites, or alternatively, to eliminatepseudosites that may serve as unwanted off-target sequences.

This report describes a general methodology for introducing recombinasetarget sequences in eukaryotic genomes using PE, the applications ofwhich are nearly limitless. The genome editing reactions are intendedfor use with “prime editor,” a chimeric fusion of a CRISPR/Cas9 proteinand a reverse-transcriptase domain, which utilizes a custom primeediting guide RNA (PEgRNA). By extension, Cas9 tools andhomology-directed repair (HDR) pathways may also be exploited tointroduce recombinase targets through DNA templates by lowering therates of indels using several techniques⁶⁵⁻⁶⁷. A proof-of-conceptexperiment in human cell culture is shown in FIG. 65 .

TABLE 6 Examples of genetic diseases linked to large-scale genomicmodifications. Disease Source Cause Trisomy 17p 68 Gene duplicationCharcot-Marie-Tooth 69 Gene duplication Smith-Magenis syndrome 70 Genedeletion Williams-Beuren 21 Gene deletion De la Chapelle syndrome 71Chromosomal translocation Down syndrome (some 72 Chromosomaltranslocation Hemophilia A 73 Gene inversion Hunter syndrome 74 Geneinversion

TABLE 7 Tyrosine recombinases and SSR target sequences. cRecombinaseSource Target Name Cre 75 ATAACTTCGTATAGCATACATTATACG loxPAAGTTAT (SEQ ID NO: 517) Dre 76 TAACTTTAAATAATGCCAATTATTTAA roxAGTTA (SEQ ID NO: 518) VCre 77 TCAATTTCTGAGAACTGTCATTCTCGG loxVAAATTGA (SEQ ID NO: 519) SCre 77 CTCGTGTCCGATAACTGTAATTATCGG loxSACATGAT (SEQ ID NO: 520) Flp 78 GAAGTTCCTATTCTCTAGAAAGTATAG FRTGAACTTC (SEQ ID NO: 521) B2 79 GAGTTTCATTAAGGAATAACTAATTCC loxBCTAATGAAACTC (SEQ ID NO: 522) B3 79 GGTTGCTTAAGAATAAGTAATTCTTAA loxB3GCAACC (SEQ ID NO: 523) Kw 80 ACGAAAAATGGTAAGGAATAGACCATTCCTTACCATTTTTGGT (SEQ ID NO: 524) R 81 TTGATGAAAGAATAACGTATTCTTTCA RSTCAA (SEQ ID NO: 525) TD1-40 82 GTGCGTCAAATAATAACGTATTATTTG TDRSACACTT (SEQ ID NO: 526) Vika 83 AATAGGTCTGAGAACGCCCATTCTCAG voxACGTATT (SEQ ID NO: 527) Nigri 84 TGAATGTCCTATAATTACACTTATAGG noxACATTCA (SEQ ID NO: 528) Panto 84 GAAACTTTAAATAATAAGTCTTATTTA poxAAGTTTC (SEQ ID NO: 529) Kd 79 AAACGATATCAGACATTTGTCTGATA loxKATGCTTCATTATCAGACAAATGTCTG ATATCGTTT (SEQ ID NO: 530) Fre 85ATATATACGTATATAGACATATATACG loxH TATATAT (SEQ ID NO: 531) CreALSHG 86ATAACTCTATATAATGTATGCTATATA loxM7 GAGTTAT (SEQ ID NO: 532) Tre 87ACAACATCCTATTACACCCTATATGCC loxLTR AACATGG (SEQ ID NO: 533) Brec1 12AACCCACTGCTTAAGCCTCAATAAAGC loxBTR TTGCCTT (SEQ ID NO: 534) Cre-R3M3 88GATACAACGTATATACCTTTCTATACG loxK2 TTGTTTA (SEQ ID NO: 535)

TABLE 8 Large serine integrases and SSR target sequences. IntegraseSource Left Target Right Target Bxb1 89 GGTTTGTCTGGTCAACCACCGGCTTGTCGACGACGGCGG GCGGTCTCAGTGGTGTACGG TCTCCGTCGTCAGGATCATTACAAACC (SEQ ID NO: 536) (SEQ ID NO: 537) phiC31 90GTGCCCCAACTGGGGTAACC TGCGGGTGCCAGGGCGTGC TTTGAGTTCTCTCAGTTGGGCCTTGGGCTCCCCGGGCGCG GG (SEQ ID NO: 538) TACTCC (SEQ ID NO: 539) R4 91TGTTCCCCAAAGCGATACCA GCATGTTCCCCAAAGCGATA CTTGAAGCAGTGGTACTGCTCCACTTGAAGCAGTGGTACT TGTGGGTACA (SEQ ID NO: GCTTGTGGGTACACTCTGCG 540)GGTG (SEQ ID NO: 541) phiBT1 92 GGTGCTGGGTTGTTGTCTCTCAGGTTTTTGACGAAAGTGA GGACAGTGATCCATGGGAA TCCAGATGATCCAG (SEQ IDACTACTCAGCACC (SEQ ID NO: 543) NO: 542) MJ1 93 ATTTTAGGTATATGATTTTGTCAAAGGATCACTGAATCAA (phiFC1) TTATTAGTGTAAATAACACT AAGTATTGCTCATCCACGCGATGTACCTAAAAT (SEQ ID AAA (SEQ ID NO: 545) NO: 544) MR11 94TTTGTGCGGAACTACGAACA CGAAAATGTATGGAGGCAC GTTCATTAATACGAAGTGTATTGTATCAATATAGGATGTA CAAACTTCCATACAA (SEQ TACCTTCGAAGACACTT ID NO: 546)(SEQ ID NO: 547) TP901-1 95 GAGTTTTTATTTCGTTTATTT ATGCCAACACAATTAACATCCAATTAAGGTAACTAAAAA TCAATCAAGGTAAATGCTTT ACTCCTTTTAAGG (SEQ IDTTGCTTTTTTTGC (SEQ ID NO: 548) NO: 549) A118 96 TTCCTCGTTTTCTCTCGTTGGTTTCGGATCAAGCTATGAAG AAGAAGAAGAAACGAGAAA GACGCAAAGAGGGAACTAA(SEQ ID NO: 550) A (SEQ ID NO: 551) U153 97 TTCCTCGTTTTCTCTCGTTGGTTTCGGATCAAGCTATGAAG ACGGAAACGAATCGAGAAA GACGCAAAGAGGGAACTAA(SEQ ID NO: 552) A (SEQ ID NO: 553) phiRV1 98 GTAGTGTATCTCACAGGTCCGAAGGTGTTGGTGCGGGGT ACGGTTGGCCGTGGACTGCT TGGCCGTGGTCGAGGTGGGGAAGAACATTCC (SEQ ID GT (SEQ ID NO: 555) NO: 554) phi370.1 99AAAAAAATACAGCGTTTTTC TTGTAAAGGAGACTGATAA ATGTACAACTATACTAGTTGTGGCATGTACAACTATACTC TAGTGCCTAAAA (SEQ ID GTCGGTAAAAAGGCA (SEQ NO: 556)ID NO: 557) TG1 100 TCCAGCCCAACAGTGTTAGT GATCAGCTCCGCGGGCAAGCTTTGCTCTTACCCAGTTGG ACCTTTCTCCTTCACGGGGT GCGGGA (SEQ ID NO: 558)GGAAGGTC (SEQ ID NO: 559) WB 101 CTAGTTTTAAAGTTGGTTATCGGAAGGTAGCGTCAACGA TAGTTACTGTGATATTTATC TAGGTGTAACTGTCGTGTTTACGGTACCCAATAACCAATG GTAACGGTACTTCCAACAGC AAT (SEQ ID NO: 560)TGGCGCCGCCAC (SEQ ID NO: 561) BL3 102 CAATGAAAAACTAGGCATGTTTTCCACAGACAACTCACGT AGAAGTTGTTTGT (SEQ ID GGAGGTAGTCAC (SEQ ID NO: 562)NO: 563) SprA 103 TGTAGTAAGTATCTTAATAT CACCCATTGTGTTCACAGGAACAGCTTTATCTGTTTTTTAA GATACAGCTTTATCTGTACT GATACTTACTACTTT (SEQ IDGATATTAATGACATGCTG NO: 564) (SEQ ID NO: 565) phiJoe 104AGTTGTGGCCATGTGTCCAT ATCTGGATGTGGGTGTCCAT CTGGGGGCAGATGGAGACGCTGCGGGCAGACGCCGCAG GGGTCACA (SEQ ID NO: 566) TCGAAGCACGG (SEQ ID NO:567) phiK38 105 CCCTAATACGCAAGTCGATA GAGCGCCGGATCAGGGAGTACTCTCCTGGGAGCGTTGAC GGACGGCCTGGGAGCGCTA AACTTGCGCACCCTGATCTGCACGCTGTGGCTGCGGTCGG (SEQ ID NO: 569) TGC (SEQ ID NO: 570) Int2 105GCTCATGTATGTGTCTACGC GGACGGCGCAGAAGGGGAG GAGATTCTCGCCCGAGAACTTAGCTCTTCGCCGGACCGTC TCTGCAAGGCACTGCTCTTG GACATACTGCTCAGCTCGTCGCT (SEQ ID NO: 571) (SEQ ID NO: 572) Int3 105 ATGGATAAAAAAATACAGCGTTTGTAAAGGAGACTGAT GTTTTTCATGTACAACTATA AATGGCATGTACAACTATACCTAGTTGTAGTGCCTAAATA TCGTCGGTAAAAAGGCATCT ATGCTT (SEQ ID NO: 573)TAT (SEQ ID NO: 574) Int4 105 AAAAATTACAAAGTTTTCAA TTCCAAAGAGCGCCCAACGCCCTTGATTTGAATTAGCGG CGACCTGAAATTTGAATAA TCAAATAATTTGTAATTCGTGACTGCTGCTTGTGTAAAGG TT (SEQ ID NO: 575) CGATGATT (SEQ ID NO: 576) Int7105 GTGTTATAAACCTGTGTGAG AGACGAGAAACGTTCCGTC AGTTAAGTTTACATGCCTAACGTCTGGGTCAGTTGGGCAA CCTTAACTTTTACGCAGGTT AGTTGATGACCGGGTCGTCCCAGCTT(SEQ ID NO: 577) GTT (SEQ ID NO: 578) Int8 105TTAATAAACTATGGAAGTAT CAATCATCAGATAACTATGG GTACAGTCTTGCAATGTTGACGGCACGTGCATTAACCAC GTGAACAAACTTCCATAATA GGTTGTATCCCGTCTAAAGTAAAT (SEQ ID NO: 579) ACTCGT (SEQ ID NO: 580) Int9 105GTGGTTGTTTTTGTTGGAAG TTTATATTGCGAAAAATAAT TGTGTATCAGGTATCTGCATTGGCGAACGAGGTAACTGG AGTTATTCCGAACTTCCAAT ATACCTCATCCGCCAATTAATA (SEQ ID NO: 581) AATTTG (SEQ ID NO: 582) Int10 105GGAAAATATAAATAATTTTA AGCACGCTGATAATCAGCA GTAACCTACATCTCAATCAAAGACCACCAACATTTCCACC GGATAGTAAAACTCTCACTC AATGTAAAAGCTTTAACCTTTT (SEQ ID NO: 583) AGC (SEQ ID NO: 584) Int11 105 GTTTATATGTTTACTAATAAATGGATTTTGCAGATTCCCA GACGCTCTCAACCCATAAAG GATGCCCCTACAGAAAGAGTCTTATTAGTAAACATATTT GTACAAAACATTTATTGGAA CAACT (SEQ ID NO: 585)TTAATT (SEQ ID NO: 586) Int12 105 TTTTTGTATGTTAGTTGTGTCGTTCGTGGTAACTATGGGTG ACTGGGTAGACCTAAATAGT GTACAGGTGCCACATTAGTTGACACAACTGCTATTAAAAT GTACCATTTATGTTTATGTG TTAA (SEQ ID NO: 587)GTTAAC (SEQ ID NO: 588) Int13 105 CAATAACGGTTGTATTTGTAGCATACATTGTTGTTGTTTT GAACTTGACCAGTTGTTTTA TCCAGATCCAGTTGGTCCTGGTAACATAAATACAACTCCG TAAATATAAGCAATCCATGT AATA (SEQ ID NO: 589)GAGT (SEQ ID NO: 590) LI 106 GTTTAGTATCTCGTTATCTCT TAACTTTTTCGGATCGAGTTCGTTGGAGGGAGAAGAAAC ATGATGGACGTAAAGAGGG GGGATACCAAAA (SEQ IDAACAAAGCATCTA (SEQ ID NO: 591) NO: 592) Peaches 107 TAGTTTCCAATGTTACAGGACGGTCTCCATCGGGATCTGC ACTGCTGGCAGAATCCAACA TGATCGAGCAGCATGCCGACATTGGAAGTCG (SEQ ID CCA (SEQ ID NO: 594) NO: 593) Bxz2 107TAACCGCAAGTGTACATCCC CGGTCTCCATCGGGATCTGC TCGGCTGGCCGAGACAAGTATGATCGAGCAGCATGCCGA CAGTTGCGACAG (SEQ ID CCA (SEQ ID NO: 596) NO: 595)SV1 108 ATGTGGTCCTTTAGATCCAC CATCAGGGCGGTCAGGCCG TGACGTGGGTCAGTGTCTCTTAGATGTGGAAGAAACGGC AAAGGACTCGCG (SEQ ID AGCACGGCGAGGACG (SEQ NO: 597)ID NO: 598)

TABLE 9 Serine resolvases and SSR target sequences. Resolvase SourceLeft Target Right Target Gin 109 CGTTTCCTGTAAACCGAGGTCGTTTCCTGTAAACCGAGGT TTTGGATAAACA (SEQ ID TTTGGATAATGG (SEQ ID NO: 599)NO: 600) Cin 110 GAGTTCTCTTAAACCAAGGT GAGTTCTCTTAAACCAAGGTTTAGGATTGAAA (SEQ ID ATTGGATAACAG (SEQ ID NO: 601) NO: 602) Hin 111TGGTTCTTGAAAACCAAGGT AAATTTTCCTTTTTGGAAGG TTTTGATAAAGC (SEQ ID NO:TTTTTGATAACCA (SEQ ID 603) NO: 604) Min 112 GCCTTCCCCTAAACCAACGTGCCTTCCCCCAAACCAAGGT TTTTATGCCGCC (SEQ ID NO: AATCAAGAACGC (SEQ ID 605)NO: 606) Sin 113 TTGTGAAATTTGGGTACACC CGTATGATTAGGGTGTATATCTAATCATACAA (SEQ ID TAATTT (SEQ ID NO: 608) NO: 607)

TABLE 10 Tyrosine integrases and target sequences. Integrase Source attPattB HK022 114 CAAATGATTTTATTTTGACTAATAA GCACTTTAGGTGATGACCTACTTACATTAATTTACTGAT AAAAGGTT (SEQ AATTAAAGAGATTTTAAATATACAAID NO: 610) CTTATTCACCTAAAGGATGACAAAA (SEQ ID NO: 609) P22 115CTAAGTGGTTTGGGACAAAAATGGG GCAGCGCATTCGT ACATACAAATCTTTGCATCGGTTTGAATGCGAAGGTCG CAAGGCTTTGCATGTCTTTCGAAGA T (SEQ ID NO: 613)TGGGACGTGTGAGCGCAGGTATGAC GTGGTATGTGTTGACTTAAAAGGTAGTTCTTATAATTCGTAATGCGAAGG TCGTAGGTTCGACTCCTATTATCGGCACCAGTTAAATCAAATACTTACGT ATTATTCGTGCCTTCCTTATTTTTACTGTGGGACATATTTGGGACAGAAGT ACCAAAAA (SEQ ID NO: 612) L5 116GCGATCCCCATCCGCGACGTGCCAA GAGCGGGCGACG CTAGGTCTCCTCTCGTCGTGAACAAGGAATCGAACCCG GGCTACCGGGTTGCAACTCCTGTGC CGTAGCTAGTTTGAACTCTCAGGCTTCAACGCGCTTCT GAAGA (SEQ ID ACGACCTGCAATTTCTTTCCACTTANO: 615) GAGGATGCAGCCGAGAGGGGTAAA AACCTATCTTGACCGGCCCATATGTGGTCGGCAGACACCCATTCTTCCAA ACTAGCTACGCGGGTTCGATTCCCGTCGCCCGCTCCGCTGGTCAGAGGGT GTTTTCGCCCTCTGGCCATTTTTCTTTCCAGGGGTCTGCAACTCTTGTGCG ACTCTTCTGACCTGGGCATACGCGGTTGCAACGCATCCCTGATCTGGCTA CTTTCGATGCTGACAAACGAATAGAGCCCCCCGCCTGCGCGAACAGACG AGGGGCATTCACA (SEQ ID NO: 614)

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Example 18—Incorporation of 3 Toeloop in the Primer Binding Site (PBS)Improves PEgRNA Activity

In order to further improve PE activity, the inventors contemplatedadding a toeloop sequence at the 3′ end of a PEgRNA having a 3′extension arm. FIG. 71A provides an example of a generic SpCas9 PEgRNAhaving a 3′ extension arm (top molecule). The 3′ extension arm, in turn,comprises an RT template (that includes that the desired edit) and aprimer binding site (PBS) at the 3′ end of the molecule. The moleculeterminates with a poly(U) sequence comprising three U nucleobases (i.e.,5′-UUU-3′).

By contrast, the bottom portion of FIG. 71A shows the same PEgRNAmolecule as the top portion of FIG. 71A, but wherein a 9-nucleobasesequence of 5′-GAAANNNNN-3′ has been inserted between the 3′ end of theprimer binding site and the 5′ end of the terminal poly(U) sequence.This structure folds back on itself by 180° to form a “toeloop” RNAstructure, wherein the sequences of 5′-NNNNN-3′ of the 9-nucleobaseinsertion anneals with a complementary sequence in the primer bindingsite, and wherein the 5′-GAAA-3′ portion forms the 180° turn. Thefeatures of the toeloop sequence depicted in FIG. 71A is not intended tolimit or narrow the scope of possible toeloops that could be used in itsplace. Further, the sequence of the toeloop will depend upon thecomplementary sequence of the primer binding site. Essentially though,the toeloop sequence, in various embodiments, may have a first sequenceportion that forms a 180°, and a second sequence portion that has asequence that is complementary to a portion of the primer binding site.

Without being bound by theory, the toeloop sequence is thought to enablePEgRNA the use of PEgRNAs with increasingly longer primer binding sitesthan would otherwise be possible. Longer PBS sequences, in turn, arethought to improve PE activity. PEgRNA More in particular, the likelyfunction of the toeloop is to occlude or at least minimize the PBS frominteracting with the spacer. Stable hairpin formation between the PBSand the spacer can lead to an inactive PEgRNA. Without a toeloop, thisinteraction may require restricting the length of the PBS. Blocking orminimizing the interaction between the spacer and the PBS using a 3′ endtoeloop may lead to an improvement in PE activity.

Example 19—Prime Editing with Alternative Nucleic Acid Templates andEditor Protein Constructs

Prior to this example, prime editing is described as requiring a PEgRNA.Exemplary embodiments describing possible configurations of suitablePEgRNA for use in prime editing are depicted in FIG. 3A (a PEgRNA with a5′ extension arm), FIG. 3B (a PEgRNA with a 3′ extension arm), FIG. 3C(an internally extended PEgRNA), FIG. 3D (a PEgRNA with a 3′ extensionarm, and comprising a primer binding site, edit template, homology arm,and optional 3′ and 5′ modifier regions, and a region indicated as theDNA synthesis template), and FIG. 3E (a PEgRNA with a 5′ extension arm,and comprising a primer binding site, edit template, homology arm, andoptional 3′ and 5′ modifier regions, and a region indicated as the DNAsynthesis template). In addition, PEgRNA structure and composition aredescribed extensively herein in the Detailed Description and throughout.

This Example describes additional design variations of PEgRNAs—in somecases, PEgRNAs which are wholly or partially chemically synthesizedoutside the cell—that are envisioned to work in conjunction with theprime editors of this Specification. Such alternative designs mayimprove various aspects of prime editing, including the insertion oflonger DNA sequences by prime editing, the use of alternativepolymerases (i.e., alternatives to reverse transcriptase) thatpotentially operate with increased efficiency and/or fidelity, and theuse or recruitment of alternative and/or addition prime editor proteineffector components to enhance or augment prime editing. In addition,the use of chemically synthesized PEgRNAs may potentially lead to theproduction of molecules that are more stable and possess desirablefeatures that enhance prime editing efficiency and capabilities.

PEgRNA serves as the nucleic acid template that encodes the desirededited genetic information that is to be incorporated into a targetsite. In one aspect, a PEgRNA is created by adding an extension arm toeither the 5′ end or 3′ end of an sgRNA (e.g., as shown in theembodiments of FIG. 3A, 3B, 3D, or 3E), or by inserting a similarsequence internally within an sgRNA (e.g., as shown in the embodiment ofFIG. 3C), wherein the extension arm comprises a DNA synthesis templatethat is capable of encoding a ssDNA product by a polymerase (e.g., areverse transcriptase) and which includes the edited genetic informationof interest. The extension arm comprises a primer binding site (PBS) forannealing to the napDNAbp-nicked genomic DNA strand, and a DNA synthesistemplate to encode t the edited DNA strand of interest, which becomesincorporated into the endogenous DNA target site by replacing thecounterpart DNA strand. PEgRNA can be expressed within cells fromplasmid DNA or a genomically integrated DNA cassette, or they can bemade outside of cells by in vitro transcription or by chemical synthesisand subsequently delivered into cells. Preparation of PEgRNAs outside ofcells, particularly by chemical synthesis, offers an opportunity tomodify the PEgRNAs substantially. This invention describes alternativedesigns for prime editing templates (FIG. 72 ).

(A) DNA Synthesis Template Expressed as a Separate Molecules from GuideRNAs (i.e., DNA Synthesis Template Provided in Trans to the Prime EditorComplex (napDNAbp+Guide RNA).

In various embodiments described herein, prime editing utilizes a singlePEgRNA that serves as both the programmable targeting molecules and theedit-encoding molecule. This embodiment is depicted in FIG. 72(a) with aPEgRNA having a 3′ extension arm. However, in some cases, this could bedisadvantageous, particularly for more complex PEgRNA molecules such asthose that encode a large insertion. These RNAs could contain extensivesecondary structure that interferes with the PEgRNA scaffold structureand interactions with Cas9. Alternatively, prime editing can be carriedout by substituting a PEgRNA with two separate RNA molecules: an sgRNA,and a trans prime editing RNA template (tPERT), as depicted in FIG. 72B.The sgRNA serves to target Cas9 (or more generally, the napDNAbp) to thedesired genomic target site, while the tPERT is used by the polymerase(e.g., a reverse transcriptase) to write new DNA sequence into thetarget locus.

In general, simple expression of a tPERT leads to lower editingefficiency compared to a PEgRNA. However, the efficiency of trans primeediting can be enhanced by the introduction of one or more MS2 RNAaptamers into the tPERT RNA, along with a fusion of the MS2 coat protein(MS2cp) to the prime editor protein (to make MS2cp-Cas9-RT). This allowsfor the MS2 RNA aptamer to bind to the MS2cp, thereby co-localizing thetPERT (which comprises the DNA synthesis template) to the site ofediting by the prime editor complex. The MS2 aptamer is preferablyplaced on the 3′ end of the tPERT to avoid reverse transcription of theaptamer sequence.

Although this example utilizes the MS2 tagging technique (comprising theMS2 RNA aptamer on the tPERT paired with the MS2cp protein fused to theprime editor), other RNA-protein recruitment systems can be used in thealternative. The general concept envisioned here is that the DNAsynthesis template of the tPERT is modified to contain an RNArecruitment secondary structure (e.g., a specialized hairpin like theMS2 aptamer) so that the tPERT may be recruited by a modified primeeditor fusion protein that further comprises an RNA-binding protein thatspecifically recognizes and binds to the RNA recruitment secondstructure on the tPERT molecule. A review of other RNA-proteinrecruitment domains are described in the art, for example, in Johanssonet al., “RNA recognition by the MS2 phage coat protein,” Sem Virol.,1997, Vol. 8(3): 176-185; Delebecque et al., “Organization ofintracellular reactions with rationally designed RNA assemblies,”Science, 2011, Vol. 333: 470-474; Mali et al., “Cas9 transcriptionalactivators for target specificity screening and paired nickases forcooperative genome engineering,” Nat. Biotechnol., 2013, Vol. 31:833-838; and Zalatan et al., “Engineering complex synthetictranscriptional programs with CRISPR RNA scaffolds,” Cell, 2015, Vol.160: 339-350, each of which are incorporated herein by reference intheir entireties. Other systems include the PP7 hairpin, whichspecifically recruits the PCP protein, and the “com” hairpin, whichspecifically recruits the Com protein. See Zalatan et al. Any of thesewell-known recruitment systems may be employed with trans prime editingas described herein.

The efficiency of the instant tPERT trans prime editing system wastested. Up to 20% efficiency of His6 insertion (18 bp) was achieved atthe HEK3 site in HEK293T cells using a tPERT containing a single 3′ MS2aptamer, a 13-nt primer binding site, and an RT template containing theinsertion sequence and 34 nt of homology to the locus, along with aneditor containing the MS2cp fused to the N-terminus of PE2. See FIG. 73. The strategy of trans prime editing has the potential to addresscomplications associated with PEgRNAPEgRNA design, and could be moresuited for longer RT templates to achieve larger insertion, deletions,or edits at further distances from the prime editor nick site.

(B) Chemically Synthesized PEgRNAs with RNA and DNA Synthesis Templates

Alternative nucleic acid templates can be used within chemicallysynthesized PEgRNAs (FIG. 72C). For example, a synthetic PEgRNA can beconstructed as an RNA/DNA hybrid wherein the spacer sequence and sgRNAscaffold is composed of RNA nucleotides and the primer binding site andsynthesis template (shown as a 3′ extension in FIG. 72C) is composed ofDNA nucleotides. This could allow for DNA-dependent DNA polymerases tobe used in place of reverse transcriptase within prime editors. It couldalso prevent the synthesis of DNA that is templated by sgRNA scaffoldsequence. In other designs, chemical linkers, composed of non-templatingnucleotides or other suitable linker moieties, can be used to tether thenucleic acid edit template (composed of RNA or DNA) to the sgRNAscaffold. This could prevent continued DNA polymerization of the sgRNAscaffold and allow for flexibility in the extension that allows for moreefficient templated synthesis. Finally, the directionality of thenucleic acid synthesis template can be inverted such that DNApolymerization proceeds away from the sgRNA scaffold as opposed totoward it.

(C) Recruitment of the DNA Polymerase Expressed in Trans

In the main embodiment of prime editing, the polymerase (e.g., reversetranscriptase enzyme) is expressed as a fusion to the napDNAbp (e.g.,Cas9 nickase). Alternatively, the polymerase (e.g., reversetranscriptase) can be expressed in trans, and its activity can belocalized to the editing site using recruitment systems such as the MS2RNA aptamer and MS2 coat protein, or other similar recruitment systemknown in the art. In this system, the PEgRNA is modified to include anMS2 aptamer within one of the sgRNA scaffold hairpins, and thepolymerase (e.g., reverse transcriptase) is expressed as a fusionprotein to MS2cp. The napDNAbp (e.g., Cas9 nickase) is also expressed asan independent polypeptide. This system has been demonstrated with thewild type M-MLV reverse transcriptase (FIG. 74 ), and should beapplicable to other RT variants. In addition, other RNA-proteininteractions, or protein-protein interactions, could be used for RTrecruitment.

The following sequences are pertinent to Example 19:

Sequences of tPERTs: MS2 aptamer/RT template/PBS/ Linker5′-MS2_13nt-PBS: (SEQ ID NO: 762) 5′GCCAACATGAGGATCACCCATGTCTGCAGGGCCTGGAGGAAGCAGGGC TTCCTTTCCTCTGCCATCAATGATGGTGATGATGGTGCGTGCTCAGTCT G - 3′5′-MS2_17nt-PBS: (SEQ ID NO: 773) 5′GCCAACATGAGGATCACCCATGTCTGCAGGGCCTGGAGGAAGCAGGGC TTCCTTTCCTCTGCCATCAATGATGGTGATGATGGTGCGTGCTCAGTCTGGGCC - 3′ 3′-MS2_13nt-PBS: (SEQ ID NO: 774)5′GGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCAATGATGGTGATGATG GTGCGTGCTCAGTCTGAAATTAACAAATCAAGCCAACATGAGGATCACCC ATGTCTGCAGGGCC - 3′ 3′-MS2_17nt-PBS:(SEQ ID NO: 775) 5′GGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCAATGATGGTGATGATGGTGCGTGCTCAGTCTGGGCC AAATTAACAAATCAAGCCAACATGAGGATCACCCATGTCTGCAGGGCC - 3′ Sequences of MS2 PEgRNAs: Spacer/MS2 aptamer/sgRNA scaffold/RT template/PBS HEK3_MS2_1 (SEQ ID NO: 776)5′ GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAG GCCAACATGAGGATCACCCATGTCTGCAGGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC TCTGCCATCTCGTGCTCAGTC T - 3′ HEK3_MS2_2(SEQ ID NO: 777) 5′ GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG GCCAACATGAGGATCACCCATGTCT GCAGGGCCAAGTGGGACCGAGTCGGTCC TCTGCCATCTCGTGCTCAGTC T - 3′ Protein sequences:MS2cp-PE2 (SEQ ID NO: 778)MKRTADGSEFESPKKKRKVGSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSGGSSGGSSGSETPGTSESATPESSGGSSGGSSDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV MS2cp-MMLV-RT (SEQ ID NO: 779)MKRTADGSEFESPKKKRKVGSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV MMLV-RT-MS2cp (SEQ ID NO: 780)MKRTADGSEFESPKKKRKVTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSGGSKRTADGSEFEPKKKRKV

Example 20. Split-Intein Delivery of Prime Editors

This Example demonstrates that a prime editor may be split using inteinsas a means to deliver the prime editor to cells via separate vectors,wherein each vector encodes a portion of the prime editor fusionprotein. This Example is focused on a PE fusion protein comprising thecanonical SpCas9 (SEQ ID NO: 18). The split sites for other Cas9proteins may be the corresponding same site, or may need to be optimizedfor each different Cas9 protein. In the instant Example, the primeeditor was split between residues 1023 and 1024 of SpCas9 (SEQ ID NO:18). This is referred to as the “1023/1024” split site.

Prime editors (PEs) exceed the AAV packaging capacity. It was thereforecontemplated to split the PE by inserting a trans-splicing Npu intein atS. pyogenes Cas9 residue 1024 in SEQ ID NO: 18, allowing delivery ofsplit-SpPEs as two separate polypeptides, each encoded by one of adual-AAV system. The split site 1023/1024 was chosen because it (1)allows packaging into two AAVs while accommodating space for guidecassette(s) and minimal regulatory elements, (2) mutation of the nativeSerine to Cysteine would be relatively conservative, (3) the site is aflexible loop near the periphery of Cas9 which sterically is predictedto allow for spicing to occur, and (4) Cas9 has been successfullyaltered in this loop by circular permutation (but not previously at thisspecific split site).

To determine whether Npu-split prime editors are active, HEK cells weretransfected with plasmids encoding split-editors, finding theyrecapitulate activity of full-length PE3 as analyzed by high-throughputsequencing. Furthermore, the three native Npu amino-terminal residues ofC-terminal extein are known to splice most efficiently but differ fromthose natively flanking Cas9 residue 1024. Replacement of these Cas9residues with the native Npu residues may alter the activity of primeeditor. It was therefore determined whether mutation from theCas9-native “SEQ” towards the Npu intein “CFN” sequence alteredefficiency of prime editing. The “SEQ” residues facilitate prime editingwith similar efficiency to full-length, suggesting the intein-split PEhalves are able to associate and mediate prime editing. No furtherincrease was seen by mutation towards “CFN”, suggesting that associationalone may be sufficient for split-PE activity, as we have previouslyobserved with intein-split base editors.

Although associated, unspliced editors may be active, perturbations ofthe sterics of the prime editor resulting from incomplete splicingduring initiation steps may affect editing outcomes. 1024-CFN wastherefore chosen for further studies as it also recapitulatesfull-length PE3 activity.

The following are the amino acid sequences of the 1023/1024 split.

SpPE2 split at 1023/1024 N terminal half (SEQ ID NO: 3875)MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY

KRTADGSEFEPKKKRKV

SpPE2 split at 1023/1024 C terminal half SEQ ID NO: 3876)

FNEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT

FPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPD

Prime Editor Packages in a Dual AAV System

Prime editors split between residues 1023 and 1024 with Nputrans-splicing intein become packages within AAV with high titercomparable to that of base editors of similar genome size produced inthe same manner.

Prime Editing In Vivo

Split SpPE3 delivered with AAV9 by intracerebroventricular injection toP0 mice mediates prime editing in brain tissue in vivo. A nucleotidesubstitution at position +5 (i.e., 5 nucleotide bases downstream of nicksite and within the PAM sequence) of a G-to-T results in theinstallation of a Pro>Gln coding mutation near the N terminus of DNMT1.This edit demonstrates the viability of prime editing in vivo and is notthought to introduce any selective pressure on edited cells (with “+5”referring to the +5 position downstream of the nick site). The AAVarchitectures tested include a full-length MMLV RT as well as atruncated variant lacking the RNAse H domain. The truncatedpost-transcriptional regulatory element W3 was also assessed as it hasbeen shown to increase expression from viral cassettes in vivo but itsimportance had not been tested in the context of base editors or primeeditors. It was found that full-length RT outperforms truncated RT, andthe addition of the W3 sequence improves activity. The increase inediting activity when W3 is present indicates that expression of primeeditor is limiting in vivo and demonstrates the distinct challenges ofprime editing in vivo versus cell culture.

REFERENCES

-   Oakes, B. L., Fellmann, C. et al. CRISPR-Cas9 Circular Permutants as    Programmable Scaffolds for Genome Modification. Cell. 2019 Jan. 10;    176(1-2):254-267.e16. doi: 10.1016/j.cell.2018.11.052.

Example 21. Linker Optimization in PE2 Format

This Example constructed a number of variant prime editors all based onPE2. PE2 has the following sequence and structure:

As used herein, “PE2” refers to a PE complex comprising a fusion proteincomprising Cas9(H840A) and a variant MMLV RT having the followingstructure:[NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]+adesired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQID NO: 134, which is shown as follows:

(SEQ ID NO: 134) MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRICRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSSGGSSGSET PGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGG SKRTADGSEFEPKKKRKVKEY: NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124), BOTTOM: (SEQ ID NO: 133) CAS9(H840A) (SEQ ID NO: 137)33-AMINO ACID LINKER  (SEQ ID NO: 127)M-MLV reverse transcriptase (SEQ ID NO: 139).The PE2 linker is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127).

In this experiment, the linker of PE2 was replaced with one of thefollowing substitute linkers (or no linker in one instance):

Linker Name Nucleotide Sequence Amino Acid Sequence 1x SGGSTCCGGAGGATCT (SEQ ID NO: 3880) SGGS (SEQ ID NO: 174) 2x SGGSTCCGGAGGATCTAGCGGAGGCTCC SGGSSGGS (SEQ ID NO: 446) (SEQ ID NO: 3881)3x SGGS TCCGGAGGATCTAGCGGAGGCTCCA SGGSSGGSSGGS (SEQ ID NO:GCGGAGGCAGC (SEQ ID NO: 3882) 3889) 1x XTEN TCTGGCTCTGAGACACCTGGCACAAGSGSETPGTSESATPES (SEQ ID CGAGAGCGCAACACCTGAAAGC NO: 171)(SEQ ID NO: 3883) No linker 1x Gly GGT G 1x Pro CCC P 1xGAAGCAGCTGCTAAA (SEQ ID NO: EAAAK (SEQ ID NO: 3968) EAAAK 3884) 2xGAAGCCGCTGCTAAAGAAGCTGCAG EAAAKEAAAK (SEQ ID NO: EAAAKCTAAG (SEQ ID NO: 3885) 3969) 3x GAAGCCGCTGCTAAAGAGGCCGCTGEAAAKEAAAKEAAAK (SEQ ID EAAAK CTAAAGAAGCTGCAGCTAAG (SEQ ID NO: 3970)NO: 3886)

FIG. 79 shows the editing efficiency of the replacement linkerconstructs. In particular, the data shows the editing efficiency of thePE2 construct with the current linker (noted as PE2—white box) comparedto various versions with the linker replaced with a sequence asindicated at the HEK3, EMX1, FANCF, RNF2 loci for representative PEgRNAsfor transition, transversion, insertion, and deletion edits. Thereplacement linkers are referred to as “1×SGGS” (SEQ ID NO:174),“2×SGGS” (SEQ ID NO: 446), “3×SGGS” (SEQ ID NO: 3889), “1×XTEN” (SEQ IDNO: 171), “no linker”, “1×Gly”, “1×Pro”, “1×EAAAK” (SEQ ID NO: 3968),“2×EAAAK” (SEQ ID NO: 3969), and “3×EAAAK” (SEQ ID NO: 3970). Theediting efficiency is measured in bar graph format relative to the“control” editing efficiency of PE2. The linker of PE2 isSGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All editing was donein the context of the PE3 system, i.e., which refers the PE2 editingconstruct plus the addition of the optimal secondary sgRNA nickingguide.

FIG. 80 . shows that the 1×XTEN (SEQ ID NO: 171) linker provided anincrease in editing efficiency. Taking the average fold efficacyrelative to PE2 yields the graph shown, indicating that use of a 1×XTEN(SEQ ID NO: 171) linker sequence improves editing efficiency by 1.14fold on average (n=15).

Example 22. Prime Editor Guide RNAs with Improved Activities

In various embodiments, PEgRNA are likely to be target DNA site and editdependent. That is, the sequence of the PEgRNA will depend upon thetarget DNA sequence and the particular edit that is being introducedtherein by prime editing (e.g., deletion, insertion, inversion,substitution). For instance, in certain embodiments, when attaching amotif 3′ of the primer binding site (PBS) of the PEgRNA, a linkerbetween the PBS and motif is preferred to prevent steric clash with thepolymerase domain (e.g., reverse transcriptase) of the PE fusionprotein. However, the nature of that linker may be different for eachsite. For instance, if the same linker for each site were used, it couldartificially render a 13 nt PBS a 16 nt PBS by fortuitous pairing to thespacer sequence. Similarly, the linker could basepair to the PBS itself,resulting in its occlusion and potentially reducing activity. So, unlikewith protein-based editors, such as PE or BE4, a single linker sequenceoption connecting two elements may not be effective in each constructbut will depend in part on the sequence of the target DNA sequence andthe edit of interest.

Building, in part, on the information presented Example 15 (Design andEngineering of PEgRNAs), this Example constructed and then tested theeffects of various structural modifications made to PEgRNA on editingfunction, among other aspects.

Expression of PEgRNAs from Non-Pol III Promoters

A variety of PEgRNA expression systems were tested for their ability togenerate PEgRNAs, using insertion of a 102 nucleotide sequence from FKBPas a readout.

Transcription of PEgRNA can be directed by a typical constitutivepromoter, such as U6 promoter. Although the U6 promoter is in most caseseffective at directing transcription of PEgRNAs, the U6 promoter is notvery effective at directing the transcription of longer PEgRNAs orU-rich RNAs. U-rich RNA stretches of cause premature termination oftranscription. This Example compared editing outcomes of guidesexpressed from the CMV promoter or U1 promoter with the U6 promoter.These promoters require a different terminator sequence, such as MASCENE or PAN ENE, as provided below. An increase in editing was observedwith the pCMV/MASC-ENE system, however these guides resulted inincomplete insertion of the sequence, while, with the U6 promoter,complete insertion was observed at lower levels of editing. See FIG. 81. The data suggests the likelihood that the alternate expression systemsmay be useful for long insertions.

The nucleotide sequence of the pCMV/MASC-ENE expression systems asfollows (5′-to-3′ direction) (with the name of the motif in boldimmediately preceding the region to which it refers):

(SEQ ID NO: 3971) -pCMV promoter-TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATC-Csy4 loop-GTTCACTGCCGTATAGGCAG-spacer-GGCCCAGACTGAGCACGTGA-scaffold-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC-template-TGGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCA-insert-AAATTTCTTTCCATCTTCAAGCATCCCGGTGTAGTGCACCACGCAGGTCTGGCCGCGCTTGGGGAAGGTGCGCCCGTCTCCTGGGGAGATGGTTTCCACCTGCACTCC-PBS-CGTGCTCAGTCTG-linker-TTT-MASC ENE-TAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGATGCTGGTGGTTGGCACTCCTGGTTTCCAGGACGGGGTTCAAATCCCTGCGGCGTCTTTGCTTTGACT-unrelated plasmid sequence-TTTTTTTAAGCTTGGGCCGCTCGAGGTAGCAGC-Ubc promoter-GGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGGAGCGTTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTTGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCACAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTAAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGGTTTGTCGTCTGGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGACAGGATCCCCGGGTACCGGTCGCCACC-Csy4 and NLS-ATGGGCTCTTTTACTATGGACCACTACCTGGATATTAGACTGAGACCTGACCCTGAGTTCCCACCCGCCCAGCTGATGAGCGTGCTGTTCGGCAAGCTGCACCAGGCCCTGGTGGCACAGGGAGGCGACCGGATCGGCGTGAGCTTCCCCGACCTGGATGAGAGCAGATCCAGGCTGGGAGAGCGCCTGAGGATCCACGCATCCGCCGACGATCTGCGCGCCCTGCTGGCCCGGCCATGGCTGGAGGGCCTGCGCGACCACCTGCAGTTTGGAGAGCCAGCAGTGGTGCCACACCCTACCCCATACAGGCAGGTGTCCAGGGTGCAGGCAAAGTCTAACCCTGAGCGGCTGCGGAGAAGGCTGATGCGCCGGCACGATCTGTCTGAGGAGGAGGCCAGAAAGAGGATCCCCGACACCGTGGCCAGAACACTGGATCTGCCTTTCGTGACCCTGCGGAGCCAGAGCACAGGCCAGCACTTCAGACTGTTTATCAGGCACGGCCCACTGCAGGTGACAGCCGAGGAAGGAGGATTCACTTGTTACGGACTGTCTAAAGGAGGATTCGTGCCCTGGTTCAGCAGCCTGAGGCCTCCTAAGAAGAAGAGGAAGGTTTAA-SV40 terminator-TGATCATAATCAAGCCATATCACATCTGTAGAGGTTTACTTGCTTTAAAAAACCTCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGC Key:[pCMV promoter] - binds pol II RNA polymerase[Csy4 loop] - bound by Csy4 protein, results incleavage 3′ of the loop. Required because part of[CMV promoter] is transcribed, and if this sequenceis attached 5′ of the gRNA it will lower/eliminateactivity (previously known). [Spacer sequence] of pegRNA[pegRNA scaffold] [DNA synthesis template][insertion edit (108 nt from FKBP)] [primer binding site][Linker](highly variable) - connects PBS and terminator element[MASC ENE transcription terminator] - transcriptionof this element results in termination of transcription;a polyA tail is encoded and then sequestered by the ENE element[Unimportant sequence][Ubc promoter] - required for expression of the Csy4 protein[Csy4 protein and NLS] - required for processing ofthe 5′ end of the guide. Other strategies could alsobe used that don't require expression of a large protein(such as ribozyme-mediated cleavage of the spacer), butthese would require more individual tuning for different spacer sequences, so we used this.[SV40 terminator] - for termination of the Csy4 protein.

Improvements to the PEgRNA Scaffold

A number of structural modifications to the gRNA scaffold were alsotested, none of which showed a significant increase in editing activity(see FIG. 82 at 3.30.13 through 3.30.19 0i the X axis, as compare to3.30). However, this data has two caveats worth noting. First, thisguide already worked quite well, and a less effective guide would havebeen better to test. Second, in HEK cells, transfection is quiteefficient, and it was noted that the amount of guide RNA transfected isin large excess compared to what is needed (reducing the amount by ˜4-8fold has no effect on editing). These improvements might only be seen inother cell types, where transfection efficiency is lower, or with lesseffective guides. Many of these changes are precedented to improve sgRNAactivity in other cell lines.

The sequences of the constructs of FIG. 82 are as follows:

HEK3.30 pegRNA sequence: [SEQ ID NO: 3972]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Templateand PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.30.0 pegRNA sequence: [SEQ ID NO: 3874]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUUUHEK3.30.1 pegRNA sequence: [SEQ ID NO: 3973]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-[none]HEK3.30.2 pegRNA sequence: [SEQ ID NO: 3974]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUGCUCGAGGCGGAAACGCCUCGAGCUUUU HEK3.30.2b pegRNA sequence:[SEQ ID NO: 3975] spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif- UUUGCUCGAGGCGGAAACGCCUCGAGCHEK3.30.3 pegRNA sequence: [SEQ ID NO: 3976]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUGCUCGAGGCGUACGCGAAAGCGUACGCCUCGAGCUUUU HEK3.30.3b pegRNA sequence:[SEQ ID NO: 3977] spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUGCUCGAGGCGUACGCGAAAGCGUACGCCUCGAGC HEK3.30.5 pegRNA sequence:[SEQ ID NO: 3978] spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUGCUCGAGGCGUACGCCCGAUGAAAAUCGGGCGUACGCCUCGAGCUU UUHEK3.30.5a pegRNA sequence: [SEQ ID NO: 3979]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUGGGGUUGGGGUUGGGGUUGGGGUUUU HEK3.30.5b pegRNA sequence:[SEQ ID NO: 3980] spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif- UUUGGUGGUGGUGGUUUUHEK3.30.13 pegRNA sequence: [SEQ ID NO: 3981]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGCGAAAGCUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.30.15 pegRNA sequence: [SEQ ID NO: 3982]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGCUCGAAAGAGCUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template andPBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.30.15 pegRNA sequence: [SEQ ID NO: 3983]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGCUCAUGAAAAUGAGCUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template andPBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.30.16 pegRNA sequence: [SEQ ID NO: 3984]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGCUCAUCCGAAAGGAUGAGCUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Templateand PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif- UUUUHEK3.30.17 pegRNA sequence: [SEQ ID NO: 3985]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGCUCAUCCUGGAAACAGGAUGAGCUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG- Terminal motif-UUUUHEK3.30.18 pegRNA sequence: [SEQ ID NO: 3986]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUGAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.30.19 pegRNA sequence: [SEQ ID NO: 3987]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUGAGAGCUAGCUCAUGAAAAUGAGCUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template andPBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.56 pegRNA sequence: [SEQ ID NO: 3890]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAAAGCUUCGACCGUGCUCAGUCUG-Terminal motif- UUUUHEK3.56.1a pegRNA sequence: [SEQ ID NO: 3891]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGGCGAAAGCCUCGUGCUCAGUCUG-Terminal motif- UUUUHEK3.56.1b pegRNA sequence: [SEQ ID NO: 3988]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGACGAAAGCCUCGUGCUCAGUCUG-Terminal motif- UUUUHEK3.56.1c pegRNA sequence: [SEQ ID NO: 3892]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGGCGAAAGCCCGUGCUCAGUCUG-Terminal motif- UUUUHEK3.56.2a pegRNA sequence: [SEQ ID NO: 3893]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGAUGCGAAAGCAUCUCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.56.2b pegRNA sequence: [SEQ ID NO: 3894]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGAUGCGAAAGCACCUCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.56.2c pegRNA sequence: [SEQ ID NO: 3895]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGAUGCGAAAGCAUCCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.56.3a pegRNA sequence: [SEQ ID NO: 3896]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGACAUGCGAAAGCAUGUCUCGUGCUCAGUCUG- Terminal motif-UUUUHEK3.56.3b pegRNA sequence: [SEQ ID NO: 3897]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGACAUGCGAAAGCAGGCCCGUGCUCAGUCUG-Terminal motif-UUUUHEK3.56.3c pegRNA sequence: [SEQ ID NO: 3898]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-UCUGCCAUCAGACAUGCGAAAGCAUGUCUCGUGCUCAGUCUG- Terminal motif-UUUUHEK3.56.4a pegRNA sequence: [SEQ ID NO: 3899]spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUUACGAAGUGGGACCGAGUCGGUCC-Template and PBS-UCUGCCAUCAAAGCUUCGACCGUGCUCAGUCUG-Terminal motif- UUUUHEK3.56.4b pegRNA sequence: [SEQ ID NO: 3989]5′motif-GCAGACCUAAGUGGUGACAUAUGGUCUG-spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUUACGAAGUGGGACCGAGUCGGUCC-Template and PBS-- Terminal motif-UUUUHEK3.56.4c pegRNA sequence: [SEQ ID NO: 3900]5′motif-GCAGACCUAAGUGGUGACAUAUGGUCUG-spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUUACGAAGUGGGACCGAGUCGGUCC-Template and PBS-- Terminal motif-UUUUHEK3.56.4d pegRNA sequence: [SEQ ID NO: 3901]5′motif-GCAGACCUAAGUGGUGACAUAUGGUCUG-spacer-GGCCCAGACUGAGCACGUGA-scaffold-GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUUACGAAGUGGGACCGAGUCGGUCC-Template and PBS-- Terminal motif-UUUUNote that where ever either no terminal motif or a terminal motif thatdoes not end in a run of U's exists, transcript was terminated using thefollowing HDV ribozyme:

[SEQ ID NO: 3990] GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGCGAAUGGGAC

Additional RNA Motifs

See FIG. 82 for details on certain motifs, such as an HDV ribozyme 3′ ofthe PEgRNA, or G-quadruplex insertion, P1 extensions, template hairpins,and tetraloop circ'd, that may be introduced into a PEgRNA to improveits performance.

In particular, this Example tested the effect of installing a tRNA motif3′ of the primer binding site. This element was chosen because ofmultiple potential functions:

-   -   (1) the tRNA motif is a very stable RNA motif, and so could        potentially reduce PEgRNA degradation;    -   (2) the MMLV RT uses a prolyl-tRNA as a primer when converting        the viral genome into DNA during transcription, so it was        suspected the same cap could be bound by the RT, improving        binding of the PEgRNA by PE, RNA stability, and bringing the PBS        back in closer proximity to the genomic site, potentially also        improving activity.

In these constructs, the P1 of the tRNA (see FIG. 84 ) was extended. P1refers to the first stem/base-pairing element of the tRNA (see FIG. 84). This was believed to be necessary to prevent RNAseP-mediated cleavageof the tRNA 5′ of the P1, which would result in its removal from thePEgRNA.

In this design a prolyl-tRNA (codon CGG) with an extended P1 and short 3nt linker between the tRNA and the PBS was used. A variety of tRNAdesigns were tested and the editing efficiency was tested compared to aPEgRNA having no tRNA cap—see the comparative data in FIG. 83 (depictinga PE experiment that targeted editing of the HEK3 gene, specificallytargeting the insertion of a 10 nt insertion at position +1 relative tothe nick site and using PE3), FIG. 85 (depicting a PE experiment thattargeted editing of the FANCF gene, specifically targeting a G-to-Tconversion at position +5 relative to the nick site and using PE3construct) and FIG. 86 (depicting a PE experiment that targeted editingof the HEK3 gene, specifically targeting the insertion of a 71 nt FLAGtag insertion at position +1 relative to the nick site and using PE3construct). tRNA-modified PEgRNAs were tested against a non-modifiedPEgRNA control.

UGG/CGG refers to the codon used, the number refers to the length of theadded P1 extension, long indicates an 8 nt linker, no designation a 3 ntlinker.

The data suggest that the installation of a tRNA may enable use ofshorter PBSs, which would likely result in additional activityimprovements. In the case of RNF2, it is possible/likely that the linkerused resulted in improved PBS binding to the spacer, and the resultingdiminishment in activity.

Some sequences used:

HEK3 +1 FLAG-tag insertion, proly-tRNA{UGG} P1 ext 5 nt, linker 3 nt[SEQ ID NO: 3902] GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUGUCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGC CUUUUFANCF +5 G to T proly-tRNA{CGG}  P1 ext 5 nt, linker 3 nt[SEQ ID NO: 3903] GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGAUCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGAC GAGCCCCGCCUUUUHEK3 ++1 10 nt insertion, proly-tRNA{UGG} P1 ext 5 nt, linker 3 nt[SEQ ID NO: 3904] GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCCUCUGCCAUCAAAGCUUCGACCGUGCUCAGUCUUCUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUU

The sequences reported in the data of FIGS. 85 and 86 are as follows:

FANCF +5 G to T pegRNA sequence: [SEQ ID NO: 3905]space, scaffold template and PBSr-GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGA-partial CGG tRNA linker8-UCUCUCUCUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUC CCGGGUUCAAUUUUFANCF +5 G to T pegRNA sequence: [SEQ ID NO: 3906]space, scaffold template and PBSr-GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 5 linker 3-UCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUU FANCF +5 G to T pegRNA sequence:[SEQ ID NO: 3907] space, scaffold template and PBSr-GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 5 linker 8-UCUCUCUCGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUUFANCF +5 G to T pegRNA sequence: [SEQ ID NO: 3908]space, scaffold template and PBSr-GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 8 linker 3-UCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUUFANCF +5 G to T pegRNA sequence: [SEQ ID NO: 3909]space, scaffold template and PBSr-GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 8 linker 8-UCUCUCUCCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUUFANCF +5 G to T pegRNA sequence: [SEQ ID NO: 3910]space, scaffold template and PBSr-GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 11 linker 3-UCUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUUFANCF +5 G to T pegRNA sequence: [SEQ ID NO: 3911]space, scaffold template and PBSr-GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 11 linker 8-UCUCUCUCGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAG CUUUUHEK3 +1 10 nt insertion pegRNA sequence: [SEQ ID NO: 3913]space, scaffold template and PBSr-GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTG-UGG P1 ext 5 linker3-UCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUUHEK3 +1 FLAG insertion pegRNA sequence: [SEQ ID NO: 3914]space, scaffold template and PBSr-GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUG-partial CGG tRNA linker 8-UCUCUCUCUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCC GGGUUCAAUUUUHEK3 +1 FLAG insertion pegRNA sequence: [SEQ ID NO: 3915]space, scaffold template and PBSr-GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUG-UGG P1 ext 5 linker 3-UCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUUHEK3 +1 FLAG insertion pegRNA sequence: [SEQ ID NO: 3916]space, scaffold template and PBSr-GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUG-UGG P1 ext 5 linker 8-UCUCUCUCGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUUHEK3 +1 FLAG insertion pegRNA sequence: [SEQ ID NO: 3917]space, scaffold template and PBSr-GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUG-UGG P1 ext 8 linker 8-UCUCUCUCCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUUHEK3 +1 FLAG insertion pegRNA sequence: [SEQ ID NO: 3918]space, scaffold template and PBSr-GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUG-UGG P1 ext 11 linker 8-UCUCUCUCGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAG CUUUUHEK3 +1 FLAG insertion pegRNA sequence: [SEQ ID NO: 3919]space, scaffold template and PBSr-GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUG-UGG P1 ext 14 linker 8-UCUCUCUCGGUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUC GAGCACCUUUURNF2 +1 C to A pegRNA sequence: [SEQ ID NO: 3920]space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{CGG}-5-UCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCC RNF2 +1 C to A pegRNA sequence:[SEQ ID NO: 3921] space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{CGG}-8-UCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUURNF2 +1 C to A pegRNA sequence: [SEQ ID NO: 3922]space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{CGG}-11-UCUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUURNF2 +1 C to A pegRNA sequence: [SEQ ID NO: 3923]space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{Lys}-5-UCUGGCGGGCCCGGAUAGCUCAGUCGGUAGAGCAUCAGACUUUUAAUCUGAGGGUCCAGGGUUCAAGUCCCUGUUCGGGCCCGCCUUUU RNF2 +1 C to A pegRNA sequence:[SEQ ID NO: 3924] space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{Lys}-8-UCUCGAGGCGGGCCCGGAUAGCUCAGUCGGUAGAGCAUCAGACUUUUAAUCUGAGGGUCCAGGGUUCAAGUCCCUGUUCGGGCCCGCCUCGUUUURNF2 +1 C to A pegRNA sequence: [SEQ ID NO: 3925]space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{UGG}-8-UCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUURNF2 +1 C to A pegRNA sequence: [SEQ ID NO: 3926]space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{UGG}-11-UCUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUURNF2 +1 C to A pegRNA sequence: [SEQ ID NO: 3927]space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{UGG}-8-longerlinker-UCUCUCUCCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCU CGUUUURNF2 +1 C to A pegRNA sequence: [SEQ ID NO: 3928]space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{UGG}-11-longerlinker-UCUCUCUCGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCG CCUCGAGCUUUURNF2 +1 C to A pegRNA sequence: [SEQ ID NO: 3929]space, scaffold template and PBSr-GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAACACCUCAUGUAAUGACUAAGAUG-tRNA-Pro{UGG}-14-longerlinker-UCUCUCUCGGUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCC CCGCCUCGAGCACCUUUU

Example 23. Use of Prime Editing to Correct CDKL5 and Sickle Cell Anemia

Use of PE to Design Mouse Model of CDKL5 with 1412delA Mutation

CDKL5 Deficiency Disorder (CDD) is a neurodegenerative disease mostoften caused by spontaneous mutations in the cyclin-dependentkinase-like 5 gene. Symptoms manifest in early childhood and includeseizures, irregular sleeping patterns, gastrointestinal stress, anddevelopmental delay. Some mutations which cause CDD, including 1412delA,cannot be corrected with base editing. However, prime editing has thepotential to precisely correct all base-to-base changes, deletions, andinsertions. With focus on the 1412delA mutation, this Example designedand tested pegRNAs capable of inserting the mutation in hopes toestablish a mouse neuronal cell line (N2A) harboring the mutation. Thiswill allow for extensive screening potentially therapeutic pegRNAs thatcorrect the mutation. The ultimate goal is to be able to move into a CDDmouse model with the 1412delA mutation to assess therapeutic effect. Nocurrent mouse models of CDD have a humanized allele, howeveroptimization of pegRNAs is underway in HEK293T cells as well. FIGS. 87and 88 are the results from a pilot screen in N2A cells where the pegRNAinstalls 1412Adel, with details about the primer binding site (PBS)length and reverse transcriptase (RT) template length. (Shown with andwithout indels)

Use of PE to Treat Sickle Cell Anemia (SCA)

Sickle cell anemia (SCA) is a recessive blood disorder caused by aglutamate-to-valine mutation at position 6 in the β-globin gene. Theresult is sickled red blood cells that are poor oxygen transporters andprone to aggregation. Symptoms of aggregation can be life-threatening.Previously, the D. Liu lab was able to show both the installation andcorrection of the SCA locus in HEK293T cells using prime editing via DNAplasmid transfection. Since hematopoietic stem cells (HSCs) aredifficult to edit via DNA plasmid transfection, this Example tested thePE3 system in HSCs with protein and mRNA nucleofection. FIG. 89 are theresults of editing at a proxy locus in the β-globin gene and at HEK3 inhealthy HSCs, varying the concentration of editor to pegRNA and nickinggRNA.

mRNA Nucleofection Protocol:

The protocol was improved by adjusting the ratio of editor to guides([editor] to [guide] ratio or editor:guide ratio)

The nicking guide protospacer sequence was:

(SEQ ID NO: 3991) CCTTGATACCAACCTGCCCA

The pegRNA sequence was:

(SEQ ID NO: 3992) CATGGTGCACCTGACTCCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAGACTTCTCTTCAGGAGTCAGGTGCACTTT

1. Thaw CD34+ cells and pre-stimulate in X-Vivo15 media with cytokines(SCF, Flt3 and TPO) for 24 hrs. Seeding density 1 million cells/mL.

2. Next day (post 24 hrs), aspirate CD34+ cells (1×105) and wash oncewith PBS (centrifugation performed at 300 g for 10 mins in RT) andresuspend in P3 solution (Lonza) (20 ul per condition).

3. In hood, combine 2 ug of SpPE2 mRNA, 1 ug combined of pegRNA andsgRNA in a sterile PCR strip on ice. Adjust the volume to 20 ul (cellsin P3 solution+mRNA &gRNA mix).

4. Pipette up cell suspension into 20 uL Lonza4D 16 well strip andelectroporate with program DS130.

5. Wait for 10 to 15 mins post electroporation and add 80 uL ofX-Vivo10+cytokine media to cell suspension and transfer to pre-warmedX-Vivo10 with cytokine media.

6. Harvest cells 72 hours after electroporation and check the cellrecovery and do genotyping in bulk population and sorted CD34+ andCD34+90+ populations as well.

Wildtype CDKL5 protein (Accession No. NP_001032420) (isoform 1 - human)(SEQ ID NO: 3993) MKIPNIGNVM NKFEILGVVG EGAYGVVLKC RHKETHEIVAIKKFKDSEEN EEVKETTLRE LKMLRTLKQE NIVELKEAFRRRGKLYLVFE YVEKNMLELL EEMPNGVPPE KVKSYIYQLIKAIHWCHKND IVHRDIKPEN LLISHNDVLK LCDFGFARNLSEGNNANYTE YVATRWYRSP ELLLGAPYGK SVDMWSVGCILGELSDGQPL FPGESEIDQL FTIQKVLGPL PSEQMKLFYSNPRFHGLRFP AVNHPQSLER RYLGILNSVL LDLMKNLLKLDPADRYLTEQ CLNHPTFQTQ RLLDRSPSRS AKRKPYHVESSTLSNRNQAG KSTALQSHHR SNSKDIQNLS VGLPRADEGLPANESFLNGN LAGASLSPLH TKTYQASSQP GSTSKDLTNNNIPHLLSPKE AKSKTEFDFN IDPKPSEGPG TKYLKSNSRSQQNRHSFMES SQSKAGTLQP NEKQSRHSYI DTIPQSSRSPSYRTKAKSHG ALSDSKSVSN LSEARAQIAE PSTSRYFPSSCLDLNSPTSP TPTRHSDTRT LLSPSGRNNR NEGTLDSRRTTTRHSKTMEE LKLPEHMDSS HSHSLSAPHE SFSYGLGYTSPFSSQQRPHR HSMYVTRDKV RAKGLDGSLS IGQGMAARANSLQLLSPQPG EQLPPEMTVA RSSVKETSRE GTSSFHTRQKSEGGVYHDPH SDDGTAPKEN RHLYNDPVPR RVGSFYRVPSPRPDNSFHEN NVSTRVSSLP SESSSGTNHS KRQPAFDPWKSPENISHSEQ LKEKEKQGFF RSMKKKKKKS QTVPNSDSPDLLTLQKSIHS ASTPSSRPKE WRPEKISDLQ TQSQPLKSLRKLLHLSSASN HPASSDPRFQ PLTAQQTKNS FSEIRIHPLSQASGGSSNIR QEPAPKGRPA LQLPDGGCDG RRQRHHSGPQDRRFMLRTTE QQGEYFCCGD PKKPHTPCVP NRALHRPISSPAPYPVLQVR GTSMCPTLQV RGTDAFSCPT QQSGFSFFVRHVMREALIHR AQVNQAALLT YHENAALTGK Wildtype CDKL5 protein(Accession No. NP_001310218) (isoform 2 - human) (SEQ ID NO: 3994)MKIPNIGNVM NKFEILGVVG EGAYGVVLKC RHKETHEIVAIKKFKDSEEN EEVKETTLRE LKMLRTLKQE NIVELKEAFRRRGKLYLVFE YVEKNMLELL EEMPNGVPPE KVKSYIYQLIKAIHWCHKND IVHRDIKPEN LLISHNDVLK LCDFGFARNLSEGNNANYTE YVATRWYRSP ELLLGAPYGK SVDMWSVGCILGELSDGQPL FPGESEIDQL FTIQKVLGPL PSEQMKLFYSNPRFHGLRFP AVNHPQSLER RYLGILNSVL LDLMKNLLKLDPADRYLTEQ CLNHPTFQTQ RLLDRSPSRS AKRKPYHVESSTLSNRNQAG KSTALQSHHR SNSKDIQNLS VGLPRADEGLPANESFLNGN LAGASLSPLH TKTYQASSQP GSTSKDLTNNNIPHLLSPKE AKSKTEFDFN IDPKPSEGPG TKYLKSNSRSQQNRHSFMES SQSKAGTLQP NEKQSRHSYI DTIPQSSRSPSYRTKAKSHG ALSDSKSVSN LSEARAQIAE PSTSRYFPSSCLDLNSPTSP TPTRHSDTRT LLSPSGRNNR NEGTLDSRRTTTRHSKTMEE LKLPEHMDSS HSHSLSAPHE SFSYGLGYTSPFSSQQRPHR HSMYVTRDKV RAKGLDGSLS IGQGMAARANSLQLLSPQPG EQLPPEMTVARSSVKETSRE GTSSFHTRQKSEGGVYHDPH SDDGTAPKEN RHLYNDPVPR RVGSFYRVPSPRPDNSFHEN NVSTRVSSLP SESSSGTNHS KRQPAFDPWKSPENISHSEQ LKEKEKQGFF RSMKKKKKKS QTVPNSDSPDLLTLQKSIHS ASTPSSRPKE WRPEKISDLQ TQSQPLKSLRKLLHLSSASN HPASSDPRFQ PLTAQQTKNS FSEIRIHPLSQASGGSSNIR QEPAPKGRPA LQLPGQMDPG WHVSSVTRSATEGPSYSEQL GAKSGPNGHP YNRTNRSRMP NLNDLKETAL

Example 24. Use of Prime Editing to Target Pathogenic AOL1 Alleles asTreatment for Non-Diabetic Chronic Kidney Disease

This Example designed PEgRNAs that are capable of targeting pathogenicAPOL1 alleles for use with prime editing to treat or reduce thelikelihood of developing a renal disease.

End-stage kidney failure (ESKD) is a growing problem that now affectsover half a million individuals in the United States. The cost of caringfor patients with ESKD is currently over 40 billion dollars per year. Inthe U.S., the likelihood that subjects of African descent will developESKD is 4 to 5 times higher than for Americans without African ancestry.These facts are reflected in the disparity between the 12-13% of theU.S. population with African descent and the 40% of U.S. dialysispatients who are African-American. The epidemic of renal disease riskfactors, such as obesity and metabolic syndrome, suggests that themagnitude of this problem will only increase.

There are no specific therapies for the vast majority of progressivekidney diseases. Some types of chronic renal disease progression can beslowed by blood pressure control with specific agents, but nephrologistscannot accurately predict which patients will respond. Moreover, whilesuccessful treatment typically slows progression, it neither preventsdisease nor halts disease progression.

Recently it was determined that that specific genetic variants thatalter the protein sequence of APOIipoprotein-L1 (APOL1) are associatedwith progressive kidney disease. Surprisingly, APOL1 kidney diseasevariants have a major impact on multiple different types of kidneydisease including hypertension-associated end-stage renal disease(H-ESRD), focal segmental glomerulosclerosis (FSGS), and HIV-associatednephropathy (HIVAN). Individuals with these variant APOL1 alleles have a7-30 fold increased risk for kidney disease. Based on the high frequencyof these APOL1 risk alleles, more than 3.5 million African Americanslikely have the high risk APOL1 genotype. African Americans without thehigh risk genotype have little excess risk compared with Americans ofEuropean ancestry.

Despite evidence that variants in the APOL1 gene cause renal disease,very little is known about the biology of its product, APOL1, or itsrole in the kidney. APOL1 has a defined role in resistance totrypanosomes, and the G1 and G2 variants appear to have become common inAfrica because they confer protection against the forms of trypanosomesthat cause African Sleeping Sickness.

There still exists a need for therapies for kidney diseases in patientswith one or more APOL1 risk alleles, which cause great morbidity andmortality with high economic impact in this and other subjectpopulations.

This Example provides three exemplary PEgRNA design options based on aspecific exemplary target sequence that may be used with prime editingto correct APOL1 defective alleles.

PEgRNA 1

Designing PEgRNAs for APOL1 allele rs73885319 (p.S342G). This representsa G→A correction in affected individuals. The target sequence is 5-

(SEQ ID NO: 3995) GGAGTCAAGCTCACGGATGTGGCCCCTGTA(G-to-A)GCTTCTTTCTTGTGCTGGATGTAGTCTACCT-3.

The protospacer (bolded above) is AAGCTCACGGATGTGGCCCC (SEQ ID NO:3996). The selected PE comprises a SaCas9(D10A).

The primer binding sites can be:

(SEQ ID NO: 3997) GTGGCCCC (SEQ ID NO: 3998) TGTGGCCCC (SEQ ID NO: 3999)ATGTGGCCCC (SEQ ID NO: 4000) GATGTGGCCCC (SEQ ID NO: 4001) GGATGTGGCCCC(SEQ ID NO: 4002) CGGATGTGGCCCC (SEQ ID NO: 4003) ACGGATGTGGCCCC(SEQ ID NO: 4004) CACGGATGTGGCCCC (SEQ ID NO: 4005) TCACGGATGTGGCCCC(SEQ ID NO: 4006) CTCACGGATGTGGCCCC.

The RT templates can be:

(SEQ ID NO: 4007) AAGAAGCTTACA (SEQ ID NO: 4008) AAAGAAGCTTACA(SEQ ID NO: 4009) GAAAGAAGCTTACA (SEQ ID NO: 4010) AGAAAGAAGCTTACA(SEQ ID NO: 4011) AAGAAAGAAGCTTACA (SEQ ID NO: 4012) CAAGAAAGAAGCTTACA(SEQ ID NO: 4013) ACAAGAAAGAAGCTTACA (SEQ ID NO: 4014)CACAAGAAAGAAGCTTACA (SEQ ID NO: 4015) GCACAAGAAAGAAGCTTACA(SEQ ID NO: 4016) AGCACAAGAAAGAAGCTTACA (SEQ ID NO: 4017)CAGCACAAGAAAGAAGCTTACA (SEQ ID NO: 4018) CCAGCACAAGAAAGAAGCTTACA(SEQ ID NO: 4019) TCCAGCACAAGAAAGAAGCTTACA (SEQ ID NO: 4020)ATCCAGCACAAGAAAGAAGCTTACA.

The nicking template can be GCITTGATTCGTACACGAGG (SEQ ID NO: 4021).

PEgRNA 2

Designing PEgRNAs for APOL1 allele rs60910145. This represents a G-Tcorrection in affected individuals.

The protospacer is GCTGGAGGAGAAGCTAAACA. (SEQ ID NO: 4022) The selectedPE comprises SpCas9(D10A)-NG.

The primer binding sites can be:

(SEQ ID NO: 4023) GAAGCTAA (SEQ ID NO: 4024) AGAAGCTAA (SEQ ID NO: 4025)GAGAAGCTAA (SEQ ID NO: 4026) GGAGAAGCTAA (SEQ ID NO: 4027) AGGAGAAGCTAA(SEQ ID NO: 4028) GAGGAGAAGCTAA (SEQ ID NO: 4029) GGAGGAGAAGCTAA(SEQ ID NO: 4030) TGGAGGAGAAGCTAA (SEQ ID NO: 4031) CTGGAGGAGAAGCTAA(SEQ ID NO: 4032) GCTGGAGGAGAAGCTAA.

The RT template can be (cannot end in C):

(SEQ ID NO: 4033) AGAATGT (SEQ ID NO: 4034) GAGAATGT (SEQ ID NO: 4035)TGAGAATGT (SEQ ID NO: 4036) TTGAGAATGT (SEQ ID NO: 4037) GTTGAGAATGT(SEQ ID NO: 4038) TGTTGAGAATGT (SEQ ID NO: 4039) TTGTTGAGAATGT(SEQ ID NO: 4040) ATTGTTGAGAATGT (SEQ ID NO: 4041) TATTGTTGAGAATGT(SEQ ID NO: 4042) TTATTGTTGAGAATGT (SEQ ID NO: 4043) ATTATTGTTGAGAATGT(SEQ ID NO: 4044) AATTATTGTTGAGAATGT (SEQ ID NO: 4045)TAATTATTGTTGAGAATGT (SEQ ID NO: 4046) ATAATTATTGTTGAGAATGT.

The nicking templates can be:

(SEQ ID NO: 4047) CCTGTGGTCACAGTTCTTGG (SEQ ID NO: 4048)CCACAGGGCAGGGCAGCCAC.

PEgRNA 3

Designing PEgRNAs for APOL1 allele rs71785313. This represents aninsert, as follows: ATTCTCAACAA[insert: TAATTA]TAAGATTC (SEQ ID NO:4049).

The protospacer can be: TCTCAACAATAAGATTCTGC (SEQ ID NO: 4050)

The PE comprises SaKKH-PE2.

The primer binding site can be:

(SEQ ID NO: 4051) TTCTCAAC (SEQ ID NO: 4052) ATTCTCAAC (SEQ ID NO: 4053)CATTCTCAAC (SEQ ID NO: 4054) ACATTCTCAAC (SEQ ID NO: 4055) AACATTCTCAAC(SEQ ID NO: 4056) AAACATTCTCAAC (SEQ ID NO: 4057) TAAACATTCTCAAC(SEQ ID NO: 4058) CTAAACATTCTCAAC (SEQ ID NO: 4059) GCTAAACATTCTCAAC(SEQ ID NO: 4060) AGCTAAACATTCTCAAC.

The RT template can be:

(SEQ ID NO: 4061) AATCTTATAATTATT (SEQ ID NO: 4062) GAATCTTATAATTATT(SEQ ID NO: 4063) AGAATCTTATAATTATT (SEQ ID NO: 4064) CAGAATCTTATAATTATT(SEQ ID NO: 4065) GCAGAATCTTATAATTATT (SEQ ID NO: 4066)TGCAGAATCTTATAATTATT (SEQ ID NO: 4067) CTGCAGAATCTTATAATTATT(SEQ ID NO: 4068) CCTGCAGAATCTTATAATTATT (SEQ ID NO: 4069)GCCTGCAGAATCTTATAATTATT (SEQ ID NO: 4070) CGCCTGCAGAATCTTATAATTATT(SEQ ID NO: 4071) CCGCCTGCAGAATCTTATAATTATT (SEQ ID NO: 4072)TCCGCCTGCAGAATCTTATAATTATT (SEQ ID NO: 4073) GTCCGCCTGCAGAATCTTATAATTATT(SEQ ID NO: 4074) GGTCCGCCTGCAGAATCTTATAATTATT.

The nicking templates can be:

(SEQ ID NO: 4047) CCTGTGGTCACAGTTCTTGG (SEQ ID NO: 4048)CCACAGGGCAGGGCAGCCAC.

OTHER REFERENCES MENTIONED THROUGHOUT THIS DISCLOSURE

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EMBODIMENTS

The following embodiments are within the scope of the presentdisclosure. Furthermore, the disclosure encompasses all variations,combinations, and permutations of these embodiments in which one or morelimitations, elements, clauses, and descriptive terms from one or moreof the listed embodiments is introduced into another listed embodimentin this section. For example, any listed embodiment that is dependent onanother embodiment can be modified to include one or more limitationsfound in any other listed embodiment in this section that is dependenton the same base embodiment. Where elements are presented as lists,e.g., in Markush group format, each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the disclosure, or aspects ofthe disclosure, is/are referred to as comprising particular elementsand/or features, certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements and/orfeatures. It is also noted that the terms “comprising” and “containing”are intended to be open and permits the inclusion of additional elementsor steps. Where ranges are given, endpoints are included. Furthermore,unless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise.

Group 1. Embodiments 1-212

1. A fusion protein comprising a nucleic acid programmable DNA bindingprotein (napDNAbp) and a reverse transcriptase.

2. The fusion protein of embodiment 1, wherein the fusion protein iscapable of carrying out genome editing by target-primed reversetranscription in the presence of an extended guide RNA.

3. The fusion protein of embodiment 1, wherein the napDNAbp has anickase activity.

4. The fusion protein of embodiment 1, wherein the napDNAbp is a Cas9protein or variant thereof.

5. The fusion protein of embodiment 1, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

6. The fusion protein of embodiment 1, wherein the napDNAbp is Cas9nickase (nCas9).

7. The fusion protein of embodiment 1, wherein the napDNAbp is selectedfrom the group consisting of: Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2C3,and Argonaute and optionally has a nickase activity.

8. The fusion protein of embodiment 1, wherein the fusion protein whencomplexed with an extended guide RNA is capable of binding to a targetDNA sequence.

9. The fusion protein of embodiment 8, wherein the target DNA sequencecomprises a target strand and a complementary non-target strand.

10. The fusion protein of embodiment 8, wherein the binding of thefusion protein complexed to the extended guide RNA forms an R-loop.

11. The fusion protein of embodiment 10, wherein the R-loop comprises(i) an RNA-DNA hybrid comprising the extended guide RNA and the targetstrand, and (ii) the complementary non-target strand.

12. The fusion protein of embodiment 11, wherein the complementarynon-target strand is nicked to form a reverse transcriptase primingsequence having a free 3′ end.

13. The fusion protein of embodiment 2, wherein the extended guide RNAcomprises (a) a guide RNA, and (b) an RNA extension at the 5′ or the 3′end of the guide RNA, or at an intramolecular location in the guide RNA.

14. The fusion protein of embodiment 13, wherein the RNA extensioncomprises (i) a reverse transcription template sequence comprising adesired nucleotide change, (ii) a reverse transcription primer bindingsite, and (iii) optionally, a linker sequence.

15. The fusion protein of embodiment 14, wherein the reversetranscription template sequence encodes a single-strand DNA flap that iscomplementary to an endogenous DNA sequence adjacent to the nick site,wherein the single-strand DNA flap comprises the desired nucleotidechange.

16. The fusion protein of embodiment 13, wherein the RNA extension is atleast 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, atleast 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, atleast 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides,at least 14 nucleotides, at least 15 nucleotides, at least 16nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, atleast 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides,or at least 25 nucleotides in length.

17. The fusion protein of embodiment 15, wherein the single-strand DNAflap hybridizes to the endogenous DNA sequence adjacent to the nicksite, thereby installing the desired nucleotide change.

18. The fusion protein of embodiment 15, wherein the single-stranded DNAflap displaces the endogenous DNA sequence adjacent to the nick site andwhich has a free 5′ end.

19. The fusion protein of embodiment 18, wherein the endogenous DNAsequence having the 5′ end is excised by the cell.

20. The fusion protein of embodiment 18, wherein cellular repair of thesingle-strand DNA flap results in installation of the desired nucleotidechange, thereby forming a desired product.

21. The fusion protein of embodiment 14, wherein the desired nucleotidechange is installed in an editing window that is between about −4 to +10of the PAM sequence, or between about −10 to +20 of the PAM sequence, orbetween about −20 to +40 of the PAM sequence, or between about −30 to+100 of the PAM sequence, or wherein the desired nucleotide change isinstalled at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotidesdownstream of the nick site.

22. The fusion protein of embodiment 1, wherein the napDNAbp comprisesan amino acid sequence of SEQ ID NO: 18, or an amino acid sequence thatis at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acidsequence to SEQ ID NO: 18.

23. The fusion protein of embodiment 1, wherein the napDNAbp comprisesan amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NOs: 18-88,126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

24. The fusion protein of embodiment 1, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

25. The fusion protein of embodiment 1, wherein the reversetranscriptase comprises an amino acid sequence that is at least 80%,85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of anyone of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154,159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

26. The fusion protein of embodiment 1, wherein the reversetranscriptase is a naturally-occurring reverse transcriptase from aretrovirus or a retrotransposon.

27. The fusion protein of any one of the previous embodiments, whereinthe fusion protein comprises the structure NH₂-[napDNAbp]-[reversetranscriptase]-COOH; or NH₂-[reverse transcriptase]-[napDNAbp]-COOH,wherein each instance of “]-[” indicates the presence of an optionallinker sequence.

28. The fusion protein of embodiment 27, wherein the linker sequencecomprises an amino acid sequence of SEQ ID NO: 127, 165-176,446,453, and767-769.

29. The fusion protein of embodiment 14, wherein the desired nucleotidechange is a single nucleotide change, an insertion of one or morenucleotides, or a deletion of one or more nucleotides.

30. The fusion protein of embodiment 29, wherein the insert or deletionis at least 1, at least 2, at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, at least 27, at least28, at least 29, at least 30, at least 31, at least 32, at least 33, atleast 34, at least 35, at least 36, at least 37, at least 38, at least39, at least 40, at least 41, at least 42, at least 43, at least 44, atleast 45, at least 46, at least 47, at least 48, at least 49, or atleast 50.

31. An extended guide RNA comprising a guide RNA and at least one RNAextension.

32. The extended guide RNA of embodiment 1, wherein the RNA extension isposition at the 3′ or 5′ end of the guide RNA, or at an intramolecularposition in the guide RNA.

33. The extended guide RNA of embodiment 31, wherein the extended guideRNA is capable of binding to a napDNAbp and directing the napDNAbp to atarget DNA sequence.

34. The extended guide RNA of embodiment 33, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand, wherein the guide RNA hybridizes to the target strand to form anRNA-DNA hybrid and an R-loop.

35. The extended guide RNA of embodiment 31, wherein the at least oneRNA extension comprises (i) a reverse transcription template sequence,(ii) a reverse transcription primer binding site, and (iii) optionally alinker sequence.

36. The extended guide RNA of embodiment 35, wherein the RNA extensionis at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, atleast 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides,at least 19 nucleotides, at least 20 nucleotides, at least 21nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least24 nucleotides, or at least 25 nucleotides in length.

37. The extended guide RNA of embodiment 35, wherein the reversetranscription template sequence is at least 3 nucleotides, at least 4nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides inlength.

38. The extended guide RNA of embodiment 35, wherein the reversetranscription primer binding site sequence is at least 3 nucleotides, atleast 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, atleast 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, atleast 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides,at least 13 nucleotides, at least 14 nucleotides, or at least 15nucleotides in length.

39. The extended guide RNA of embodiment 35, wherein the optional linkersequence is at least 3 nucleotides, at least 4 nucleotides, at least 5nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, or at least 15 nucleotides in length.

40. The extended guide RNA of embodiment 35, wherein the reversetranscription template sequence encodes a single-strand DNA flap that iscomplementary to an endogenous DNA sequence adjacent to a nick site,wherein the single-strand DNA flap comprises a desired nucleotidechange.

41. The extended guide RNA of embodiment 40, wherein the single-strandedDNA flap displaces an endogenous single-strand DNA having a 5′ end inthe target DNA sequence that has been nicked, and wherein the endogenoussingle-strand DNA is immediately adjacent downstream of the nick site.

42. The extended guide RNA of embodiment 41, wherein the endogenoussingle-stranded DNA having the free 5′ end is excised by the cell.

43. The extended guide RNA of embodiment 41, wherein cellular repair ofthe single-strand DNA flap results in installation of the desirednucleotide change, thereby forming a desired product.

44. The extended guide RNA of embodiment 31, comprising the nucleotidesequence of SEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103,3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810, ora nucleotide sequence having at least 85%, or at least 90%, or at least95%, or at least 98%, or at least 99% sequence identity with any one ofSEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121,3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810.

45. The extended guide RNA of embodiment 35, wherein the reversetranscription template sequence comprises a nucleotide sequence that isat least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenousDNA target.

46. The extended guide RNA of embodiment 35, wherein the reversetranscription primer binding site hybridizes with a free 3′ end of thecut DNA.

47. The extended guide RNA of embodiment 35, wherein the optional linkersequence is at least 1 nucleotide, or at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, or at least 15nucleotides in length.

48. A complex comprising a fusion protein of any one of embodiments 1-30and an extended guide RNA.

49. The complex of embodiment 48, wherein the extended guide RNAcomprises a guide RNA and an RNA extension at the 3′ or 5′ end of theguide RNA or at an intramolecular position in the guide RNA.

50. The complex of embodiment 48, wherein the extended guide RNA iscapable of binding to a napDNAbp and directing the napDNAbp to a targetDNA sequence.

51. The complex of embodiment 50, wherein the target DNA sequencecomprises a target strand and a complementary non-target strand, whereinthe guide RNA hybridizes to the target strand to form an RNA-DNA hybridand an R-loop.

52. The complex of embodiment 49, wherein the at least one RNA extensioncomprises (i) a reverse transcription template sequence, (ii) a reversetranscription primer binding site, and (iii) optionally a linkersequence.

53. The complex of embodiment 48, wherein the extended guide RNAcomprises the nucleotide sequence of SEQ ID NOs: 394, 429-442, 641-649,678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556,3628-3698, and 3755-3810, or a nucleotide sequence having at least 85%,or at least 90%, or at least 95%, or at least 98%, or at least 99%sequence identity with any one of SEQ ID NOs: 394, 429-442, 641-649,678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556,3628-3698, and 3755-3810.

54. The complex of embodiment 52, wherein the reverse transcriptiontemplate sequence comprises a nucleotide sequence having at least 80%,or 85%, or 90%, or 95%, or 99% sequence identity with the endogenous DNAtarget.

55. The complex of embodiment 52, wherein the reverse transcriptionprimer binding site hybridizes with a free 3′ end of the cut DNA.

56. A complex comprising a napDNAbp and an extended guide RNA.

57. The complex of embodiment 56, wherein the napDNAbp is a Cas9nickase.

58. The complex of embodiment 56, wherein the napDNAbp comprises anamino acid sequence of SEQ ID NO: 18, or an amino acid sequence havingat least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ IDNO: 18.

59. The complex of embodiment 57, wherein the napDNAbp comprises anamino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity with the amino acid sequence of any one of SEQ ID NOs:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

60. The complex of embodiment 57, wherein the extended guide RNAcomprises a guide RNA and an RNA extension at the 3′ or 5′ end of theguide RNA, or at an intramolecular position in the guide RNA.

61. The complex of embodiment 57, wherein the extended guide RNA iscapable of directing the napDNAbp to a target DNA sequence.

62. The complex of embodiment 61, wherein the target DNA sequencecomprises a target strand and a complementary non-target strand, whereinthe spacer sequence hybridizes to the target strand to form an RNA-DNAhybrid and an R-loop.

63. The complex of embodiment 61, wherein the RNA extension comprises(i) a reverse transcription template sequence, (ii) a reversetranscription primer binding site, and (iii) optionally a linkersequence.

64. The complex of embodiment 57, wherein the extended guide RNAcomprises the nucleotide sequence of SEQ ID NOs: 394, 429-442, 641-649,678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556,3628-3698, and 3755-3810, or a nucleotide sequence having at least 85%,or at least 90%, or at least 95%, or at least 98%, or at least 99%sequence identity with any one of SEQ ID NOs: 394, 429-442, 641-649,678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556,3628-3698, and 3755-3810.

65. The complex of embodiment 63, wherein the reverse transcriptiontemplate sequence comprises a nucleotide sequence that is at least 80%,or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.

66. The complex of embodiment 63, wherein the reverse transcriptionprimer binding site hybridizes with a free 3′ end of the cut DNA.

67. The complex of embodiment 63, wherein the optional linker sequenceis at least 1 nucleotide, or at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, or at least 15nucleotides in length.

68. A polynucleotide encoding the fusion protein of any of embodiments1-30.

69. A vector comprising the polynucleotide of embodiment 68.

70. A cell comprising the fusion protein of any of embodiments 1-30 andan extended guide RNA bound to the napDNAbp of the fusion protein.

71. A cell comprising a complex of any one of embodiments 48-67.

72. A pharmaceutical composition comprising: (i) a fusion protein of anyof embodiments 1-30, the complex of embodiments 48-67, thepolynucleotide of embodiment 68, or the vector of embodiment 69; and(ii) a pharmaceutically acceptable excipient.

73. A pharmaceutical composition comprising: (i) the complex ofembodiments 48-67 (ii) reverse transcriptase provided in trans; and(iii) a pharmaceutically acceptable excipient.

74. A kit comprising a nucleic acid construct, comprising: (i) a nucleicacid sequencing encoding the fusion protein of any one of embodiments1-30; and (ii) a promoter that drives expression of the sequence of (i).

75. A method for installing a desired nucleotide change in adouble-stranded DNA sequence, the method comprising:

-   -   (i) contacting the double-stranded DNA sequence with a complex        comprising a fusion protein and an extended guide RNA, wherein        the fusion protein comprises a napDNAbp and a reverse        transcriptase and wherein the extended guide RNA comprises a        reverse transcription template sequence comprising the desired        nucleotide change;    -   (ii) nicking the double-stranded DNA sequence on the non-target        strand, thereby generating a free single-strand DNA having a 3′        end;    -   (iii) hybridizing the 3′ end of the free single-strand DNA to        the reverse transcription template sequence, thereby priming the        reverse transcriptase domain;    -   (iv) polymerizing a strand of DNA from the 3′ end, thereby        generating a single-strand DNA flap comprising the desired        nucleotide change;    -   (v) replacing an endogenous DNA strand adjacent the cut site        with the single-strand DNA flap, thereby installing the desired        nucleotide change in the double-stranded DNA sequence.

76. The method of embodiment 75, wherein the step of (v) replacingcomprises: (i) hybridizing the single-strand DNA flap to the endogenousDNA strand adjacent the cut site to create a sequence mismatch; (ii)excising the endogenous DNA strand; and (iii) repairing the mismatch toform the desired product comprising the desired nucleotide change inboth strands of DNA.

77. The method of embodiment 76, wherein the desired nucleotide changeis a single nucleotide substitution, a deletion, or an insertion.

78. The method of embodiment 77, wherein the single nucleotidesubstitution is a transition or a transversion.

79. The method of embodiment 76, wherein the desired nucleotide changeis (1) a G to T substitution, (2) a G to A substitution, (3) a G to Csubstitution, (4) a T to G substitution, (5) a T to A substitution, (6)a T to C substitution, (7) a C to G substitution, (8) a C to Tsubstitution, (9) a C to A substitution, (10) an A to T substitution,(11) an A to G substitution, or (12) an A to C substitution.

80. The method of embodiment 76, wherein the desired nucleoid changeconverts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to anA:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair toa G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:Abasepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) aC:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair,(10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:Cbasepair, or (12) an A:T basepair to a C:G basepair.

81. The method of embodiment 76, wherein the desired nucleotide changeis an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

82. The method of embodiment 76, wherein the desired nucleotide changecorrects a disease-associated gene.

83. The method of embodiment 82, wherein the disease-associated gene isassociated with a monogenetic disorder selected from the groupconsisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 AntitrypsinDeficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; MarfanSyndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease;Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.

84. The method of embodiment 82, wherein the disease-associated gene isassociated with a polygenic disorder selected from the group consistingof: heart disease; high blood pressure; Alzheimer's disease; arthritis;diabetes; cancer; and obesity.

85. The method of embodiment 76, wherein the napDNAbp is a nuclease deadCas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.

86. The method of embodiment 76, wherein the napDNAbp comprises an aminoacid sequence of SEQ ID NO: 18.

87. The method of embodiment 76, wherein the napDNAbp comprises an aminoacid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with the amino acid sequence of any one of SEQ ID NOs: 18-88,126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

88. The method of embodiment 76, wherein the reverse transcriptasecomprises any one of the amino acid sequences of SEQ ID NOs: 89-100,105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662,700, 701-716, 739-741, and 766.

89. The method of embodiment 76, wherein the reverse transcriptasedomain comprises an amino acid sequence having at least 80%, 85%, 90%,95%, 98%, or 99% sequence identity with the amino acid sequence of anyone of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154,159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

90. The method of embodiment 76, wherein the extended guide RNAcomprises an RNA extension at the 3′ or 5′ ends or at an intramolecularlocation in the guide RNA, wherein the RNA extension comprises thereverse transcription template sequence.

91. The method of embodiment 90, wherein the RNA extension is at least 5nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides,at least 17 nucleotides, at least 18 nucleotides, at least 19nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or atleast 25 nucleotides in length.

92. The method of embodiment 76, wherein the extended guide RNA has anucleotide sequence selected from the group consisting of SEQ ID NOs:394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455,3479-3493, 3522-3556, 3628-3698, and 3755-3810.

93. A method for introducing one or more changes in the nucleotidesequence of a DNA molecule at a target locus, comprising:

-   -   (i) contacting the DNA molecule with a nucleic acid programmable        DNA binding protein (napDNAbp) and a guide RNA which targets the        napDNAbp to the target locus, wherein the guide RNA comprises a        reverse transcriptase (RT) template sequence comprising at least        one desired nucleotide change;    -   (ii) forming an exposed 3′ end in a DNA strand at the target        locus;    -   (iii) hybridizing the exposed 3′ end to the RT template sequence        to prime reverse transcription;    -   (iv) synthesizing a single strand DNA flap comprising the at        least one desired nucleotide change based on the RT template        sequence by reverse transcriptase;    -   (v) and incorporating the at least one desired nucleotide change        into the corresponding endogenous DNA, thereby introducing one        or more changes in the nucleotide sequence of the DNA molecule        at the target locus.

94. The method of embodiment 93, wherein the one or more changes in thenucleotide sequence comprises a transition.

95. The method of embodiment 94, wherein the transition is selected fromthe group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) Gto A.

96. The method of embodiment 93, wherein the one or more changes in thenucleotide sequence comprises a transversion.

97. The method of embodiment 96, wherein the transversion is selectedfrom the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) Cto A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.

98. The method of embodiment 93, wherein the one or more changes in thenucleotide sequence comprises changing (1) a G:C basepair to a T:Abasepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair toC:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepairto an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:Gbasepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) aC:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair,(11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:Gbasepair.

99. The method of embodiment 93, wherein the one or more changes in thenucleotide sequence comprises an insertion or deletion of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 nucleotides.

100. The method of embodiment 93, wherein the one or more changes in thenucleotide sequence comprises a correction to a disease-associated gene.

101. The method of embodiment 100, wherein the disease-associated geneis associated with a monogenetic disorder selected from the groupconsisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 AntitrypsinDeficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; MarfanSyndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease;Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.

102. The method of embodiment 100, wherein the disease-associated geneis associated with a polygenic disorder selected from the groupconsisting of: heart disease; high blood pressure; Alzheimer's disease;arthritis; diabetes; cancer; and obesity.

103. The method of embodiment 93, wherein the napDNAbp is a nucleaseactive Cas9 or variant thereof.

104. The method of embodiment 93, wherein the napDNAbp is a nucleaseinactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variant thereof.

105. The method of embodiment 93, wherein the napDNAbp comprises anamino acid sequence of SEQ ID NO: 18.

106. The method of embodiment 93, wherein the napDNAbp comprises anamino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity with the amino acid sequence of any one of SEQ ID NOs:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

107. The method of embodiment 93, wherein the reverse transcriptase isintroduced in trans.

108. The method of embodiment 93, wherein the napDNAbp comprises afusion to a reverse transcriptase.

109. The method of embodiment 93, wherein the reverse transcriptasecomprises any one of the amino acid sequences of SEQ ID NOs: 89-100,105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662,700, 701-716, 739-741, and 766.

110. The method of embodiment 93, wherein the reverse transcriptasecomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235,454, 471, 516, 662, 700, 701-716, 739-741, and 766.

111. The method of embodiment 93, wherein the step of forming an exposed3′ end in the DNA strand at the target locus comprises nicking the DNAstrand with a nuclease.

112. The method of embodiment 111, wherein the nuclease is the napDNAbp,is provided as a fusion domain of napDNAbp, or is provided in trans.

113. The method of embodiment 93, wherein the step of forming an exposed3′ end in the DNA strand at the target locus comprises contacting theDNA strand with a chemical agent.

114. The method of embodiment 93, wherein the step of forming an exposed3′ end in the DNA strand at the target locus comprises introducing areplication error.

115. The method of embodiment 93, wherein the step of contacting the DNAmolecule with the napDNAbp and the guide RNA forms an R-loop.

116. The method of embodiment 115, wherein the DNA strand in which theexposed 3′ end is formed is in the R-loop.

117. The method of embodiment 93, wherein guide RNA comprises anextended portion that comprises the reverse transcriptase (RT) templatesequence.

118. The method of embodiment 117, wherein the extended portion is atthe 3′ end of the guide RNA, the 5′ end of the guide RNA, or at anintramolecular position in the guide RNA.

119. The method of embodiment 93, wherein the guide RNA furthercomprises a primer binding site.

120. The method of embodiment 93, wherein the guide RNA furthercomprises a spacer sequence.

121. The method of embodiment 93, wherein the RT template sequence ishomologous to the corresponding endogenous DNA.

122. A method for introducing one or more changes in the nucleotidesequence of a DNA molecule at a target locus by target-primed reversetranscription, the method comprising: (a) contacting the DNA molecule atthe target locus with a (i) fusion protein comprising a nucleic acidprogrammable DNA binding protein (napDNAbp) and a reverse transcriptaseand (ii) a guide RNA comprising an RT template comprising a desirednucleotide change; (b) conducting target-primed reverse transcription ofthe RT template to generate a single strand DNA comprising the desirednucleotide change; and (c) incorporating the desired nucleotide changeinto the DNA molecule at the target locus through a DNA repair and/orreplication process.

123. The method of embodiment 122, wherein the RT template is located atthe 3′ end of the guide RNA, the 5′ end of the guide RNA, or at anintramolecular location in the guide RNA.

124. The method of embodiment 122, wherein the desired nucleotide changecomprises a transition, a transversion, an insertion, or a deletion, orany combination thereof.

125. The method of embodiment 122, wherein the desired nucleotide changecomprises a transition selected from the group consisting of: (a) T toC; (b) A to G; (c) C to T; and (d) G to A.

126. The method of embodiment 122, wherein the desired nucleotide changecomprises a transversion selected from the group consisting of: (a) T toA; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G toC; and (h) G to T.

127. The method of embodiment 122, wherein the desired nucleotide changecomprises changing (1) a G:C basepair to a T:A basepair, (2) a G:Cbasepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) aT:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair,(6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:Cbasepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to anA:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:Tbasepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.

128. A polynucleotide encoding the extended guide RNA of any one ofembodiments 31-47.

129. A vector comprising the polynucleotide of embodiment 128.

130. A cell comprising the vector of embodiment 129.

131. The fusion protein of any of embodiments 1-30, wherein the reversetranscriptase is an error-prone reverse transcriptase.

132. A method for mutagenizing a DNA molecule at a target locus bytarget-primed reverse transcription, the method comprising: (a)contacting the DNA molecule at the target locus with a (i) fusionprotein comprising a nucleic acid programmable DNA binding protein(napDNAbp) and an error-prone reverse transcriptase and (ii) a guide RNAcomprising an RT template comprising a desired nucleotide change; (b)conducting target-primed reverse transcription of the RT template togenerate a mutagenized single strand DNA; and (c) incorporating themutagenized single strand DNA into the DNA molecule at the target locusthrough a DNA repair and/or replication process.

133. The method of embodiment 132, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

134. The method of embodiment 132, wherein the napDNAbp is a Cas9nickase (nCas9).

135. The method of embodiment 132, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

136. The method of embodiment 132, wherein the guide RNA comprises SEQID NO: 222.

137. The method of embodiment 132, wherein the step of (b) conductingtarget-primed reverse transcription comprises generating a 3′ end primerbinding sequence at the target locus that is capable of priming reversetranscription by annealing to a primer binding site on the guide RNA.

138. A method for replacing a trinucleotide repeat expansion mutation ina target DNA molecule with a healthy sequence comprising a healthynumber of repeat trinucleotides, the method comprising: (a) contactingthe DNA molecule at the target locus with a (i) fusion proteincomprising a nucleic acid programmable DNA binding protein (napDNAbp)and a reverse transcriptase and (ii) a guide RNA comprising an RTtemplate comprising the replacement sequence, wherein said fusionprotein intr; (b) conducting target-primed reverse transcription of theRT template to generate a single strand DNA comprising the replacementsequence; and (c) incorporating the single strand DNA into the DNAmolecule at the target locus through a DNA repair and/or replicationprocess.

139. The method of embodiment 138, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

140. The method of embodiment 138, wherein the napDNAbp is a Cas9nickase (nCas9).

141. The method of embodiment 138, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

142. The method of embodiment 138, wherein the guide RNA comprises SEQID NO: 222.

143. The method of embodiment 138, wherein the step of (b) conductingtarget-primed reverse transcription comprises generating a 3′ end primerbinding sequence at the target locus that is capable of priming reversetranscription by annealing to a primer binding site on the guide RNA.

144. The method of embodiment 138, wherein the trinucleotide repeatexpansion mutation is associated with Huntington's Disease, Fragile Xsyndrome, or Friedreich's ataxia.

145. The method of embodiment 138, wherein the trinucleotide repeatexpansion mutation comprises a repeating unit of CAG triplets.

146. The method of embodiment 138, wherein the trinucleotide repeatexpansion mutation comprises a repeating unit of GAA triplets.

147. A method of installing a functional moiety in a protein of interestencoded by a target nucleotide sequence by prime editing, the methodcomprising: (a) contacting the target nucleotide sequence with a (i)prime editor comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising anedit template encoding the functional moiety; (b) polymerizing a singlestrand DNA sequence encoding the functional moiety; and (c)incorporating the single strand DNA sequence in place of a correspondingendogenous strand at the target nucleotide sequence through a DNA repairand/or replication process, wherein the method produces a recombinanttarget nucleotide sequence that encodes a fusion protein comprising theprotein of interest and the functional moiety.

148. The method of embodiment 147, wherein functional moiety is peptidetag.

149. The method of embodiment 148, wherein the peptide tag is anaffinity tag, solubilization tag, chromatography tag, epitope tag, or afluorescence tag.

150. The method of embodiment 148, wherein the peptide tag is selectedfrom the group consisting of: AviTag (SEQ ID NO: 245); C-tag (SEQ ID NO:246); Calmodulin-tag (SEQ ID NO: 247); polyglutamate tag (SEQ ID NO:248); E-tag (SEQ ID NO: 249); FLAG-tag (SEQ ID NO: 2); HA-tag (SEQ IDNO: 5); His-tag (SEQ ID NOs: 252-262); Myc-tag (SEQ ID NO: 6); NE-tag(SEQ ID NO: 264); Rho1D4-tag (SEQ ID NO: 265); S-tag (SEQ ID NO: 266);SBP-tag (SEQ ID NO: 267); Softag-1 (SEQ ID NO: 268); Softag-2 (SEQ IDNO: 269); Spot-tag (SEQ ID NO: 270); Strep-tag (SEQ ID NO: 271); TC tag(SEQ ID NO: 272); Ty tag (SEQ ID NO: 273); V5 tag (SEQ ID NO: 3);VSV-tag (SEQ ID NO: 275); and Xpress tag (SEQ ID NO: 276).

151. The method of embodiment 148, wherein the peptide tag is selectedfrom the group consisting of: AU1 epitope (SEQ ID NO: 278); AU5 epitope(SEQ ID NO: 279); Bacteriophage T7 epitope (T7-tag) (SEQ ID NO: 280);Bluetongue virus tag (B-tag) (SEQ ID NO: 281); E2 epitope (SEQ ID NO:282); Histidine affinity tag (HAT) (SEQ ID NO: 283); HSV epitope (SEQ IDNO: 284); Polyarginine (Arg-tag) (SEQ ID NO: 285); Polyaspartate(Asp-tag) (SEQ ID NO: 286); Polyphenylalanine (Phe-tag) (SEQ ID NO:287); S1-tag (SEQ ID NO: 288); S-tag (SEQ ID NO: 266); and VSV-G (SEQ IDNO: 275).

152. The method of embodiment 147, wherein the functional moiety is animmunoepitope.

153. The method of embodiment 152, wherein the immunoepitope is selectedfrom the group consisting of: tetanus toxoid (SEQ ID NO: 396);diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1(SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumpsimmunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406);hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1(SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes towardclass I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA(SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO:420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422);hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO:424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ IDNO: 426).

154. The method of embodiment 147, wherein the functional moiety altersthe localization of the protein of interest.

155. The method of embodiment 147, wherein the functional moiety is adegradation tag such that the degradation rate of the protein ofinterest is altered.

156. The method of embodiment 155, wherein the degradation tag comprisesan amino acid sequence encoding the degradation tags as disclosedherein.

157. The method of embodiment 147, wherein the functional moiety is asmall molecule binding domain.

158. The method of embodiment 157, wherein the small molecule bindingdomain is FKBP12 of SEQ ID NO: 488.

159. The method of embodiment 157, wherein the small molecule bindingdomain is FKBP12-F36V of SEQ ID NO: 489.

160. The method of embodiment 157, wherein the small molecule bindingdomain is cyclophilin of SEQ ID NOs: 490 and 493-494.

161. The method of embodiment 157, wherein the small molecule bindingdomain is installed in two or more proteins of interest.

162. The method of embodiment 161, wherein the two or more proteins ofinterest may dimerize upon contacting with a small molecule.

163. The method of embodiment 157, wherein the small molecule is a dimerof a small molecule selected from the group consisting of:

164. A method of installing an immunoepitope in a protein of interestencoded by a target nucleotide sequence by prime editing, the methodcomprising: (a) contacting the target nucleotide sequence with a (i)prime editor comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising anedit template encoding the functional moiety; (b) polymerizing a singlestrand DNA sequence encoding the immunoepitope; and (c) incorporatingthe single strand DNA sequence in place of a corresponding endogenousstrand at the target nucleotide sequence through a DNA repair and/orreplication process, wherein the method produces a recombinant targetnucleotide sequence that encodes a fusion protein comprising the proteinof interest and the immunoepitope.

165. The method of embodiment 164, wherein the immunoepitope is selectedfrom the group consisting of: tetanus toxoid (SEQ ID NO: 396);diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1(SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumpsimmunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406);hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1(SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes towardclass I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA(SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO:420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422);hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO:424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ IDNO: 426).

166. The method of embodiment 164, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

167. The method of embodiment 164, wherein the napDNAbp is a Cas9nickase (nCas9).

168. The method of embodiment 164, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

169. The method of embodiment 164, wherein the guide RNA comprises SEQID NO: 222.

170. A method of installing a small molecule dimerization domain in aprotein of interest encoded by a target nucleotide sequence by primeediting, the method comprising: (a) contacting the target nucleotidesequence with a (i) prime editor comprising a nucleic acid programmableDNA binding protein (napDNAbp) and a reverse transcriptase and (ii) aPEgRNA comprising an edit template encoding the small moleculedimerization domain; (b) polymerizing a single strand DNA sequenceencoding the immunoepitope; and (c) incorporating the single strand DNAsequence in place of a corresponding endogenous strand at the targetnucleotide sequence through a DNA repair and/or replication process,wherein the method produces a recombinant target nucleotide sequencethat encodes a fusion protein comprising the protein of interest and thesmall molecule dimerization domain.

171. The method of embodiment 170, further comprising conducting themethod on a second protein of interest.

172. The method of embodiment 171, wherein the first protein of interestand the second protein of interest dimerize in the presence of a smallmolecule that binds to the dimerization domain on each of said proteins.

173. The method of embodiment 170, wherein the small molecule bindingdomain is FKBP12 of SEQ ID NO: 488.

174. The method of embodiment 170, wherein the small molecule bindingdomain is FKBP12-F36V of SEQ ID NO: 489.

175. The method of embodiment 170, wherein the small molecule bindingdomain is cyclophilin of SEQ ID NOs: 490 and 493-494.

176. The method of embodiment 170, wherein the small molecule is a dimerof a small molecule selected from the group consisting of:

177. The method of embodiment 170, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

178. The method of embodiment 170, wherein the napDNAbp is a Cas9nickase (nCas9).

179. The method of embodiment 170, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

180. The method of embodiment 170, wherein the guide RNA comprises SEQID NO: 222.

181. A method of installing a peptide tag or epitope onto a proteinusing prime editing, comprising: contacting a target nucleotide sequenceencoding the protein with a prime editor construct configured to inserttherein a second nucleotide sequence encoding the peptide tag to resultin a recombinant nucleotide sequence, such that the peptide tag and theprotein are expressed from the recombinant nucleotide sequence as afusion protein.

182. The method of embodiment 181, wherein the peptide tag is used forpurification and/or detection of the protein.

183. The method of embodiment 181, wherein the peptide tag is apoly-histidine (e.g., HHHHHH (SEQ ID NO: 252-262), FLAG (e.g., DYKDDDDK(SEQ ID NO: 2)), V5 (e.g., GKPIPNPLLGLDST (SEQ ID NO: 3)), GCN4, HA(e.g., YPYDVPDYA (SEQ ID NO: 5)), Myc (e.g. EQKLISEED(SEQ ID NO: 6)),GST . . . etc.

184. The method of embodiment 181, wherein the peptide tag has an aminoacid sequence selected from the group consisting of SEQ ID NO: 245-290.

185. The method of embodiment 181, wherein the peptide tag is fused tothe protein by a linker.

186. The method of embodiment 181, wherein the fusion protein has thefollowing structure: [protein]-[peptide tag] or [peptide tag]-[protein],wherein “]-[” represents an optional linker.

187. The method of embodiment 181, wherein the linker has an amino acidsequence of SEQ ID NO: 127, 165-176, 446, 453, and 767-769.

188. The method of embodiment 181, wherein the prime editor constructcomprises a PEgRNA comprising the nucleotide sequence of SEQ ID NOs:18-101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777,2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556,3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, 3972-3989.

189. The method of embodiment 181, wherein the PEgRNA comprises aspacer, a gRNA core, and an extension arm, wherein the spacer iscomplementary to the target nucleotide sequence and the extension armcomprises a reverse transcriptase template that encodes the peptide tag.

190. The method of embodiment 181, wherein the PEgRNA comprises aspacer, a gRNA core, and an extension arm, wherein the spacer iscomplementary to the target nucleotide sequence and the extension armcomprises a reverse transcriptase template that encodes the peptide tag.

191. A method of preventing or halting the progression of a priondisease by installing on or more protective mutations into PRNP encodedby a target nucleotide sequence by prime editing, the method comprising:(a) contacting the target nucleotide sequence with a (i) prime editorcomprising a nucleic acid programmable DNA binding protein (napDNAbp)and a reverse transcriptase and (ii) a PEgRNA comprising an edittemplate encoding the functional moiety; (b) polymerizing a singlestrand DNA sequence encoding the protective mutation; and (c)incorporating the single strand DNA sequence in place of a correspondingendogenous strand at the target nucleotide sequence through a DNA repairand/or replication process, wherein the method produces a recombinanttarget nucleotide sequence that encodes a PRNP comprising a protectivemutation and which is resistant to misfolding.

192. The method of embodiment 191, wherein the prion disease is a humanprion disease.

193. The method of embodiment 191, wherein the prion disease is ananimal prion disease.

194. The method of embodiment 192, wherein the prion disease isCreutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease(vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal FamilialInsomnia, or Kuru.

195. The method of embodiment 193, wherein the prion disease is BovineSpongiform Encephalopathy (BSE or “mad cow disease”), Chronic WastingDisease (CWD), Scrapie, Transmissible Mink Encephalopathy, FelineSpongiform Encephalopathy, and Ungulate Spongiform Encephalopathy.

196. The method of embodiment 191, wherein the wildtype PRNP amino acidsequence is SEQ ID NOs: 291-292.

197. The method of embodiment 191, wherein the method results in amodified PRNP amino acid sequence selected from the group consisting ofSEQ ID NOs: 293-323, wherein said modified PRNP protein is resistant tomisfolding.

198. The method of embodiment 191, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

199. The method of embodiment 191, wherein the napDNAbp is a Cas9nickase (nCas9).

200. The method of embodiment 191, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

201. The method of embodiment 191, wherein the guide RNA comprises SEQID NO: 222.

202. A method of installing a ribonucleotide motif or tag in an RNA ofinterest encoded by a target nucleotide sequence by prime editing, themethod comprising: (a) contacting the target nucleotide sequence with a(i) prime editor comprising a nucleic acid programmable DNA bindingprotein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNAcomprising an edit template encoding the ribonucleotide motif or tag;(b) polymerizing a single strand DNA sequence encoding theribonucleotide motif or tag; and (c) incorporating the single strand DNAsequence in place of a corresponding endogenous strand at the targetnucleotide sequence through a DNA repair and/or replication process,wherein the method produces a recombinant target nucleotide sequencethat encodes a modified RNA of interest comprising the ribonucleotidemotif or tag.

203. The method of embodiment 202, wherein ribonucleotide motif or tagis a detection moiety.

204. The method of embodiment 202, wherein the ribonucleotide motif ortag affects the expression level of the RNA of interest.

205. The method of embodiment 202, wherein the ribonucleotide motif ortag affects the transport or subcellular location of the RNA ofinterest.

206. The method of embodiment 202, wherein the ribonucleotide motif ortag is selected from the group consisting of SV40 type 1, SV40 type 2,SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE,Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer,spinach aptamer, mango aptamer, HDV ribozyme, and m6A.

207. The method of embodiment 202, wherein the PEgRNA comprises SEQ IDNOs: 101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761,776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540,3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929,3972-3989.

208. The method of embodiment 202, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

209. The method of embodiment 202, wherein the napDNAbp is a Cas9nickase (nCas9).

210. The method of embodiment 202, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

211. A method of installing or deleting a functional moiety in a proteinof interest encoded by a target nucleotide sequence by prime editing,the method comprising: (a) contacting the target nucleotide sequencewith a (i) prime editor comprising a nucleic acid programmable DNAbinding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNAcomprising an edit template encoding the functional moiety or deletionof same; (b) polymerizing a single strand DNA sequence encoding thefunctional moiety or deletion of same; and (c) incorporating the singlestrand DNA sequence in place of a corresponding endogenous strand at thetarget nucleotide sequence through a DNA repair and/or replicationprocess, wherein the method produces a recombinant target nucleotidesequence that encodes a modified protein comprising the protein ofinterest and the functional moiety or the removal of same, wherein thefunctional moiety alters a modification state or localization state ofthe protein.

212. The method of embodiment 211, wherein functional moiety alters thephosphorylation, ubiquitylation, glycosylation, lipidation,hydroxylation, methylation, acetylation, crotonylation, SUMOylationstate of the protein of interest.

Group 2. Embodiments 213-424

213. A fusion protein comprising a nucleic acid programmable DNA bindingprotein (napDNAbp) and a polymerase.

214. The fusion protein of embodiment 213, wherein the fusion protein iscapable of carrying out prime editing in the presence of an primeediting guide RNA (PEgRNA).

215. The fusion protein of embodiment 213, wherein the napDNAbp has anickase activity.

216. The fusion protein of embodiment 213, wherein the napDNAbp is aCas9 protein or variant thereof.

217. The fusion protein of embodiment 213, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

218. The fusion protein of embodiment 213, wherein the napDNAbp is Cas9nickase (nCas9).

219. The fusion protein of embodiment 213, wherein the napDNAbp isselected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a,Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickaseactivity.

220. The fusion protein of embodiment 213, wherein the fusion proteinwhen complexed with a PEgRNA is capable of binding to a target DNAsequence.

221. The fusion protein of embodiment 220, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand.

222. The fusion protein of embodiment 220, wherein the binding of thefusion protein complexed to the PEgRNA forms an R-loop.

223. The fusion protein of embodiment 222, wherein the R-loop comprises(i) an RNA-DNA hybrid comprising the PEgRNA and the target strand, and(ii) the complementary non-target strand.

224. The fusion protein of embodiment 223, wherein the complementarynon-target strand is nicked to form a priming sequence having a free 3′end.

225. The fusion protein of embodiment 214, wherein the PEgRNA comprises(a) a guide RNA and (b) an extension arm at the 5′ or the 3′ end of theguide RNA, or at an intramolecular location in the guide RNA.

226. The fusion protein of embodiment 225, wherein the extension armcomprises (i) a DNA synthesis template sequence comprising a desirednucleotide change, and (ii) a primer binding site.

227. The fusion protein of embodiment 226, wherein the DNA synthesistemplate sequence encodes a single-strand DNA flap that is complementaryto an endogenous DNA sequence adjacent to the nick site, wherein thesingle-strand DNA flap comprises the desired nucleotide change.

228. The fusion protein of embodiment 225, wherein the extension arm isat least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides,at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides,at least 11 nucleotides, at least 12 nucleotides, at least 13nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, atleast 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides,at least 22 nucleotides, at least 23 nucleotides, at least 24nucleotides, or at least 25 nucleotides in length.

229. The fusion protein of embodiment 227, wherein the single-strand DNAflap hybridizes to the endogenous DNA sequence adjacent to the nicksite, thereby installing the desired nucleotide change.

230. The fusion protein of embodiment 227, wherein the single-strandedDNA flap displaces the endogenous DNA sequence adjacent to the nick siteand which has a free 5′ end.

231. The fusion protein of embodiment 230, wherein the endogenous DNAsequence having the 5′ end is excised by the cell.

232. The fusion protein of embodiment 230, wherein cellular repair ofthe single-strand DNA flap results in installation of the desirednucleotide change, thereby forming a desired product.

233. The fusion protein of embodiment 226, wherein the desirednucleotide change is installed in an editing window that is betweenabout −4 to +10 of the PAM sequence, or between about −10 to +20 of thePAM sequence, or between about −20 to +40 of the PAM sequence, orbetween about −30 to +100 of the PAM sequence, or wherein the desirednucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 nucleotides downstream of the nick site.

234. The fusion protein of embodiment 213, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18, or an amino acidsequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with the amino acid sequence to SEQ ID NO: 18.

235. The fusion protein of embodiment 213, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

236. The fusion protein of embodiment 213, wherein the polymerase is areverse transcriptase comprising any one of the amino acid sequences ofSEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235,454, 471, 516, 662, 700, 701-716, 739-741, and 766.

237. The fusion protein of embodiment 213, wherein the polymerase is areverse transcriptase comprising an amino acid sequence having at least80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acidsequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139,143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and766.

238. The fusion protein of embodiment 213, wherein the polymerase is anaturally-occurring reverse transcriptase from a retrovirus or aretrotransposon.

239. The fusion protein of any one of the previous embodiments, whereinthe fusion protein comprises the structureNH₂-[napDNAbp]-[polymerase]-COOH; or NH₂-[polymerase]-[napDNAbp]-COOH,wherein each instance of “]-[” indicates the presence of an optionallinker sequence.

240. The fusion protein of embodiment 239, wherein the linker sequencecomprises an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453,and 767-769.

241. The fusion protein of embodiment 226, wherein the desirednucleotide change is a single nucleotide change, an insertion of one ormore nucleotides, or a deletion of one or more nucleotides.

242. The fusion protein of embodiment 241, wherein the insert ordeletion is at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33, at least 34, at least 35, at least 36, at least 37, at least 38, atleast 39, at least 40, at least 41, at least 42, at least 43, at least44, at least 45, at least 46, at least 47, at least 48, at least 49, orat least 50.

243. A PEgRNA comprising a guide RNA and at least one nucleic acidextension arm comprising a DNA synthesis template.

244. The PEgRNA of embodiment 241, wherein the nucleic acid extensionarm is position at the 3′ or 5′ end of the guide RNA, or at anintramolecular position in the guide RNA, and wherein the nucleic acidextension arm is DNA or RNA.

245. The PEgRNA of embodiment 242, wherein the PEgRNA is capable ofbinding to a napDNAbp and directing the napDNAbp to a target DNAsequence.

246. The PEgRNA of embodiment 245, wherein the target DNA sequencecomprises a target strand and a complementary non-target strand, whereinthe guide RNA hybridizes to the target strand to form an RNA-DNA hybridand an R-loop.

247. The PEgRNA of embodiment 243, wherein the at least one nucleic acidextension arm comprises (i) a DNA synthesis template, and (ii) a primerbinding site.

248. The PEgRNA of embodiment 247, wherein the nucleic acid extensionarm is at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, atleast 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides,at least 19 nucleotides, at least 20 nucleotides, at least 21nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least24 nucleotides, or at least 25 nucleotides in length.

249. The PEgRNA of embodiment 247, wherein the DNA synthesis template isat least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides,at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides,at least 9 nucleotides, at least 10 nucleotides, at least 11nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least14 nucleotides, or at least 15 nucleotides in length.

250. The PEgRNA of embodiment 247, wherein the primer binding site is atleast 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, atleast 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, atleast 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides,at least 12 nucleotides, at least 13 nucleotides, at least 14nucleotides, or at least 15 nucleotides in length.

251. The PEgRNA of embodiment 243, further comprising at least oneadditional structure selected from the group consisting of a linker, astem loop, a hairpin, a toeloop, an aptamer, or an RNA-proteinrecruitment domain.

252. The PEgRNA of embodiment 247, wherein the DNA synthesis templateencodes a single-strand DNA flap that is complementary to an endogenousDNA sequence adjacent to a nick site, wherein the single-strand DNA flapcomprises a desired nucleotide change.

253. The PEgRNA of embodiment 252, wherein the single-stranded DNA flapdisplaces an endogenous single-strand DNA having a 5′ end in the targetDNA sequence that has been nicked, and wherein the endogenoussingle-strand DNA is immediately adjacent downstream of the nick site.

254. The PEgRNA of embodiment 253, wherein the endogenoussingle-stranded DNA having the free 5′ end is excised by the cell.

255. The PEgRNA of embodiment 253, wherein cellular repair of thesingle-strand DNA flap results in installation of the desired nucleotidechange, thereby forming a desired product.

256. The PEgRNA of embodiment 243, comprising the nucleotide sequence ofSEQ ID NOs: 101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334,336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362,364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736,757-761, 776-777, or a nucleotide sequence having at least 85%, or atleast 90%, or at least 95%, or at least 98%, or at least 99% sequenceidentity with any one of SEQ ID NOs: 101-104, 181-183, 223-234, 237-244,277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649,678-692, 735-736, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455,3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901,3905-3911, 3913-3929, 3972-3989.

257. The PEgRNA of embodiment 247, wherein the DNA synthesis templatecomprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or95%, or 99% identical to the endogenous DNA target.

258. The PEgRNA of embodiment 247, wherein the primer binding sitehybridizes with a free 3′ end of the cut DNA.

259. The PEgRNA of embodiment 251, wherein the at least one additionalstructure is located at the 3′ or 5′ end of the PEgRNA.

260. A complex comprising a fusion protein of any one of embodiments213-242 and an PEgRNA.

261. The complex of embodiment 260, wherein the PEgRNA comprises a guideRNA and an nucleic acid extension arm at the 3′ or 5′ end of the guideRNA or at an intramolecular position in the guide RNA.

262. The complex of embodiment 260, wherein the PEgRNA is capable ofbinding to a napDNAbp and directing the napDNAbp to a target DNAsequence.

263. The complex of embodiment 262, wherein the target DNA sequencecomprises a target strand and a complementary non-target strand, whereinthe guide RNA hybridizes to the target strand to form an RNA-DNA hybridand an R-loop.

264. The complex of embodiment 261, wherein the at least one nucleicacid extension arm comprises (i) a DNA synthesis template, and (ii) aprimer binding site.

265. The complex of embodiment 260, wherein the PEgRNA comprises thenucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-234, 237-244,277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649,678-692, 735-736, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455,3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901,3905-3911, 3913-3929, 3972-3989, or a nucleotide sequence having atleast 85%, or at least 90%, or at least 95%, or at least 98%, or atleast 99% sequence identity with any one of SEQ ID NOs: 101-104,181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394,429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777,2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556,3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, 3972-3989.

266. The complex of embodiment 264, wherein the DNA synthesis templatecomprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or95%, or 99% identical to the endogenous DNA target.

267. The complex of embodiment 264, wherein the primer binding sitehybridizes with a free 3′ end of the cut DNA.

268. A complex comprising a napDNAbp and an PEgRNA.

269. The complex of embodiment 268, wherein the napDNAbp is a Cas9nickase.

270. The complex of embodiment 268, wherein the napDNAbp comprises anamino acid sequence of SEQ ID NO: 18.

271. The complex of embodiment 268, wherein the napDNAbp comprises anamino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity with the amino acid sequence of any one of SEQ ID NOs:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

272. The complex of embodiment 268, wherein the PEgRNA comprises a guideRNA and a nucleic acid extension arm at the 3′ or 5′ end of the guideRNA, or at an intramolecular position in the guide RNA.

273. The complex of embodiment 268, wherein the PEgRNA is capable ofdirecting the napDNAbp to a target DNA sequence.

274. The complex of embodiment 272, wherein the target DNA sequencecomprises a target strand and a complementary non-target strand, whereinthe spacer sequence of the PEgRNA hybridizes to the target strand toform an RNA-DNA hybrid and an R-loop.

275. The complex of embodiment 273, wherein the nucleic acid extensionarm comprises (i) a DNA synthesis template, and (ii) a primer bindingsite.

276. The complex of embodiment 269, wherein the PEgRNA comprises thenucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-234, 237-244,277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649,678-692, 735-736, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455,3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901,3905-3911, 3913-3929, 3972-3989, or a nucleotide sequence having atleast 85%, or at least 90%, or at least 95%, or at least 98%, or atleast 99% sequence identity with any one of SEQ ID NOs: 101-104,181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394,429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777,2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556,3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, 3972-3989.

277. The complex of embodiment 276, wherein the DNA synthesis templatecomprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or95%, or 99% identical to the endogenous DNA target.

278. The complex of embodiment 276, wherein the primer binding sitehybridizes with a free 3′ end of the cut DNA.

279. The complex of embodiment 276, wherein the PEgRNA further comprisesat least one additional structure selected from the group consisting ofa linker, a stem loop, a hairpin, a toeloop, an aptamer, or anRNA-protein recruitment domain.

280. A polynucleotide encoding the fusion protein of any of embodiments213-242.

281. A vector comprising the polynucleotide of embodiment 280.

282. A cell comprising the fusion protein of any of embodiments 213-242and an PEgRNA bound to the napDNAbp of the fusion protein.

283. A cell comprising a complex of any one of embodiments 260-279.

284. A pharmaceutical composition comprising: (i) a fusion protein ofany of embodiments 213-242, the complex of embodiments 260-279, thepolynucleotide of embodiment 68, or the vector of embodiment 69; and(ii) a pharmaceutically acceptable excipient.

285. A pharmaceutical composition comprising: (i) the complex ofembodiments 260-279 (ii) a polymerase provided in trans; and (iii) apharmaceutically acceptable excipient.

286. A kit comprising a nucleic acid construct, comprising: (i) anucleic acid sequencing encoding the fusion protein of any one ofembodiments 213-242; and (ii) a promoter that drives expression of thesequence of (i).

287. A method for installing a desired nucleotide change in adouble-stranded DNA sequence, the method comprising:

-   -   (i) contacting the double-stranded DNA sequence with a complex        comprising a fusion protein and a PEgRNA, wherein the fusion        protein comprises a napDNAbp and a polymerase and wherein the        PEgRNA comprises a DNA synthesis template comprising the desired        nucleotide change and a primer binding site;    -   (ii) nicking the double-stranded DNA sequence, thereby        generating a free single-strand DNA having a 3′ end;    -   (iii) hybridizing the 3′ end of the free single-strand DNA to        the primer binding site, thereby priming the polymerase;    -   (iv) polymerizing a strand of DNA from the 3′ end hybridized to        the primer binding site, thereby generating a single-strand DNA        flap comprising the desired nucleotide change and which is        complementary to the DNA synthesis template;    -   (v) replacing an endogenous DNA strand adjacent the cut site        with the single-strand DNA flap, thereby installing the desired        nucleotide change in the double-stranded DNA sequence.

288. The method of embodiment 287, wherein the step of (v) replacingcomprises: (i) hybridizing the single-strand DNA flap to the endogenousDNA strand adjacent the cut site to create a sequence mismatch; (ii)excising the endogenous DNA strand; and (iii) repairing the mismatch toform the desired product comprising the desired nucleotide change inboth strands of DNA.

289. The method of embodiment 288, wherein the desired nucleotide changeis a single nucleotide substitution, a deletion, or an insertion.

290. The method of embodiment 289, wherein the single nucleotidesubstitution is a transition or a transversion.

291. The method of embodiment 288, wherein the desired nucleotide changeis (1) a G to T substitution, (2) a G to A substitution, (3) a G to Csubstitution, (4) a T to G substitution, (5) a T to A substitution, (6)a T to C substitution, (7) a C to G substitution, (8) a C to Tsubstitution, (9) a C to A substitution, (10) an A to T substitution,(11) an A to G substitution, or (12) an A to C substitution.

292. The method of embodiment 288, wherein the desired nucleoid changeconverts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to anA:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair toa G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:Abasepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) aC:G basepair to a T:A basepair, a C:G basepair to an A:T basepair, (10)an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:Cbasepair, or (12) an A:T basepair to a C:G basepair.

293. The method of embodiment 288, wherein the desired nucleotide changeis an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

294. The method of embodiment 288, wherein the desired nucleotide changecorrects a disease-associated gene.

295. The method of embodiment 294, wherein the disease-associated geneis associated with a monogenetic disorder selected from the groupconsisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 AntitrypsinDeficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; MarfanSyndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease;Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a priondisease; and Tay-Sachs Disease.

296. The method of embodiment 294, wherein the disease-associated geneis associated with a polygenic disorder selected from the groupconsisting of: heart disease; high blood pressure; Alzheimer's disease;arthritis; diabetes; cancer; and obesity.

297. The method of embodiment 287, wherein the napDNAbp is a nucleasedead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.

298. The method of embodiment 287, wherein the napDNAbp comprises anamino acid sequence of SEQ ID NO: 18, or an amino acid sequence havingat least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with theamino acid sequence of SEQ ID NO: 18.

299. The method of embodiment 287, wherein the napDNAbp comprises anamino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity with the amino acid sequence of any one of SEQ ID NOs:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

300. The method of embodiment 287, wherein the polymerase is a reversetranscriptase comprising any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

301. The method of embodiment 287, wherein the polymerase is a reversetranscriptase comprising an amino acid sequence having at least 80%,85%, 90%, 95%, 98%, or 99% sequence identity with the amino acidsequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139,143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and766.

302. The method of embodiment 287, wherein the PEgRNA comprises anucleic acid extension arm at the 3′ or 5′ ends or at an intramolecularlocation in the guide RNA, wherein the extension arm comprises the DNAsynthesis template sequence and the primer binding site.

303. The method of embodiment 302, wherein the extension arm is at least5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides,at least 17 nucleotides, at least 18 nucleotides, at least 19nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or atleast 25 nucleotides in length.

304. The method of embodiment 287, wherein the PEgRNA has a nucleotidesequence selected from the group consisting of SEQ ID NOs: 101-104,181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394,429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777,2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556,3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, 3972-3989.

305. A method for introducing one or more changes in the nucleotidesequence of a DNA molecule at a target locus, comprising:

-   -   (i) contacting the DNA molecule with a nucleic acid programmable        DNA binding protein (napDNAbp) and a PEgRNA which targets the        napDNAbp to the target locus, wherein the PEgRNA comprises a        reverse transcriptase (RT) template sequence comprising at least        one desired nucleotide change and a primer binding site;    -   (ii) forming an exposed 3′ end in a DNA strand at the target        locus;    -   (iii) hybridizing the exposed 3′ end to the primer binding site        to prime reverse transcription;    -   (iv) synthesizing a single strand DNA flap comprising the at        least one desired nucleotide change based on the RT template        sequence by reverse transcriptase;    -   (v) and incorporating the at least one desired nucleotide change        into the corresponding endogenous DNA, thereby introducing one        or more changes in the nucleotide sequence of the DNA molecule        at the target locus.

306. The method of embodiment 305, wherein the one or more changes inthe nucleotide sequence comprises a transition.

307. The method of embodiment 306, wherein the transition is selectedfrom the group consisting of: (a) T to C; (b) A to G; (c) C to T; and(d) G to A.

308. The method of embodiment 305, wherein the one or more changes inthe nucleotide sequence comprises a transversion.

309. The method of embodiment 308, wherein the transversion is selectedfrom the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) Cto A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.

310. The method of embodiment 305, wherein the one or more changes inthe nucleotide sequence comprises changing (1) a G:C basepair to a T:Abasepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair toC:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepairto an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:Gbasepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) aC:G basepair to an A:T basepair, (9) an A:T basepair to a T:A basepair,(11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:Gbasepair.

311. The method of embodiment 305, wherein the one or more changes inthe nucleotide sequence comprises an insertion or deletion of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 nucleotides.

312. The method of embodiment 305, wherein the one or more changes inthe nucleotide sequence comprises a correction to a disease-associatedgene.

313. The method of embodiment 312, wherein the disease-associated geneis associated with a monogenetic disorder selected from the groupconsisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 AntitrypsinDeficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; MarfanSyndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease;Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a priondisease; and Tay-Sachs Disease.

314. The method of embodiment 312, wherein the disease-associated geneis associated with a polygenic disorder selected from the groupconsisting of: heart disease; high blood pressure; Alzheimer's disease;arthritis; diabetes; cancer; and obesity.

315. The method of embodiment 305, wherein the napDNAbp is a nucleaseactive Cas9 or variant thereof.

316. The method of embodiment 305, wherein the napDNAbp is a nucleaseinactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variant thereof.

317. The method of embodiment 305, wherein the napDNAbp comprises anamino acid sequence of SEQ ID NO: 18, or an amino acid sequence havingat least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ IDNO: 18.

318. The method of embodiment 305, wherein the napDNAbp comprises anamino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity with the amino acid sequence of any one of SEQ ID NOs:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

319. The method of embodiment 305, wherein the reverse transcriptase isintroduced in trans.

320. The method of embodiment 305, wherein the napDNAbp comprises afusion to a reverse transcriptase.

321. The method of embodiment 305, wherein the reverse transcriptasecomprises any one of the amino acid sequences of SEQ ID NOs: 89-100,105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662,700, 701-716, 739-741, and 766.

322. The method of embodiment 305, wherein the reverse transcriptasecomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235,454, 471, 516, 662, 700, 701-716, 739-741, and 766.

323. The method of embodiment 305, wherein the step of forming anexposed 3′ end in the DNA strand at the target locus comprises nickingthe DNA strand with a nuclease.

324. The method of embodiment 323, wherein the nuclease is provided isprovided in trans.

325. The method of embodiment 305, wherein the step of forming anexposed 3′ end in the DNA strand at the target locus comprisescontacting the DNA strand with a chemical agent.

326. The method of embodiment 305, wherein the step of forming anexposed 3′ end in the DNA strand at the target locus comprisesintroducing a replication error.

327. The method of embodiment 305, wherein the step of contacting theDNA molecule with the napDNAbp and the guide RNA forms an R-loop.

328. The method of embodiment 327, wherein the DNA strand in which theexposed 3′ end is formed is in the R-loop.

329. The method of embodiment 315, wherein the PEgRNA comprises anextension arm that comprises the reverse transcriptase (RT) templatesequence and the primer binding site.

330. The method of embodiment 329, wherein the extension arm is at the3′ end of the guide RNA, the 5′ end of the guide RNA, or at anintramolecular position in the guide RNA.

331. The method of embodiment 305, wherein the PEgRNA further comprisesat least one additional structure selected from the group consisting ofa linker, a stem loop, a hairpin, a toeloop, an aptamer, or anRNA-protein recruitment domain.

332. The method of embodiment 305, wherein the PEgRNA further comprisesa homology arm.

333. The method of embodiment 305, wherein the RT template sequence ishomologous to the corresponding endogenous DNA.

334. A method for introducing one or more changes in the nucleotidesequence of a DNA molecule at a target locus by target-primed reversetranscription, the method comprising: (a) contacting the DNA molecule atthe target locus with a (i) fusion protein comprising a nucleic acidprogrammable DNA binding protein (napDNAbp) and a reverse transcriptaseand (ii) a guide RNA comprising an RT template comprising a desirednucleotide change; (b) conducting target-primed reverse transcription ofthe RT template to generate a single strand DNA comprising the desirednucleotide change; and (c) incorporating the desired nucleotide changeinto the DNA molecule at the target locus through a DNA repair and/orreplication process.

335. The method of embodiment 334, wherein the RT template is located atthe 3′ end of the guide RNA, the 5′ end of the guide RNA, or at anintramolecular location in the guide RNA.

336. The method of embodiment 334, wherein the desired nucleotide changecomprises a transition, a transversion, an insertion, or a deletion, orany combination thereof.

337. The method of embodiment 334, wherein the desired nucleotide changecomprises a transition selected from the group consisting of: (a) T toC; (b) A to G; (c) C to T; and (d) G to A.

338. The method of claim 334, wherein the desired nucleotide changecomprises a transversion selected from the group consisting of: (a) T toA; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G toC; and (h) G to T.

339. The method of embodiment 334, wherein the desired nucleotide changecomprises changing (1) a G:C basepair to a T:A basepair, (2) a G:Cbasepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) aT:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair,(6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:Cbasepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to anA:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:Tbasepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.

340. A polynucleotide encoding the PEgRNA of any one of embodiments243-259.

341. A vector comprising the polynucleotide of embodiment 340.

342. A cell comprising the vector of embodiment 341.

343. The fusion protein of embodiment 213, wherein the polymerase is anerror-prone reverse transcriptase.

344. A method for mutagenizing a DNA molecule at a target locus bytarget-primed reverse transcription, the method comprising: (a)contacting the DNA molecule at the target locus with a (i) fusionprotein comprising a nucleic acid programmable DNA binding protein(napDNAbp) and an error-prone reverse transcriptase and (ii) a guide RNAcomprising an RT template comprising a desired nucleotide change; (b)conducting target-primed reverse transcription of the RT template togenerate a mutagenized single strand DNA; and (c) incorporating themutagenized single strand DNA into the DNA molecule at the target locusthrough a DNA repair and/or replication process.

345. The method of any prior embodiment, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

346. The method of any prior embodiment, wherein the napDNAbp is a Cas9nickase (nCas9).

347. The method of embodiment 344, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

348. The method of embodiment 344, wherein the guide RNA comprises SEQID NO: 222.

349. The method of embodiment 344, wherein the step of (b) conductingtarget-primed reverse transcription comprises generating a 3′ end primerbinding sequence at the target locus that is capable of priming reversetranscription by annealing to a primer binding site on the guide RNA.

350. A method for replacing a trinucleotide repeat expansion mutation ina target DNA molecule with a healthy sequence comprising a healthynumber of repeat trinucleotides, the method comprising: (a) contactingthe DNA molecule at the target locus with a (i) fusion proteincomprising a nucleic acid programmable DNA binding protein (napDNAbp)and a polymerase and (ii) a PEgRNA comprising DNA synthesis templatecomprising the replacement sequence and a primer binding site; (b)conducting prime editing to generate a single strand DNA comprising thereplacement sequence; and (c) incorporating the single strand DNA intothe DNA molecule at the target locus through a DNA repair and/orreplication process.

351. The method of embodiment 350, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

352. The method of embodiment 350, wherein the napDNAbp is a Cas9nickase (nCas9).

353. The method of embodiment 350, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

354. The method of embodiment 350, wherein the guide RNA comprises SEQID NO: 222.

355. The method of embodiment 350, wherein the step of (b) conductingprime editing comprises generating a 3′ end primer binding sequence atthe target locus that is capable of priming polymerase by annealing tothe primer binding site on the guide RNA.

356. The method of embodiment 350, wherein the trinucleotide repeatexpansion mutation is associated with Huntington's Disease, Fragile Xsyndrome, or Friedreich's ataxia.

357. The method of embodiment 350, wherein the trinucleotide repeatexpansion mutation comprises a repeating unit of CAG triplets.

358. The method of embodiment 350, wherein the trinucleotide repeatexpansion mutation comprises a repeating unit of GAA triplets.

359. A method of installing a functional moiety in a protein of interestencoded by a target nucleotide sequence by prime editing, the methodcomprising: (a) contacting the target nucleotide sequence with a (i)prime editor comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a polymerase and (ii) a PEgRNA comprising DNA synthesistemplate encoding the functional moiety; (b) polymerizing a singlestrand DNA sequence encoding the functional moiety; and (c)incorporating the single strand DNA sequence in place of a correspondingendogenous strand at the target nucleotide sequence through a DNA repairand/or replication process, wherein the method produces a recombinanttarget nucleotide sequence that encodes a fusion protein comprising theprotein of interest and the functional moiety.

360. The method of embodiment 359, wherein functional moiety is peptidetag.

361. The method of embodiment 360, wherein the peptide tag is anaffinity tag, solubilization tag, chromatography tag, epitope orimmunoepitope tag, or a fluorescence tag.

362. The method of embodiment 360, wherein the peptide tag is selectedfrom the group consisting of: AviTag (SEQ ID NO: 245); C-tag (SEQ ID NO:246); Calmodulin-tag (SEQ ID NO: 247); polyglutamate tag (SEQ ID NO:248); E-tag (SEQ ID NO: 249); FLAG-tag (SEQ ID NO: 2); HA-tag (SEQ IDNO: 5); His-tag (SEQ ID NOs: 252-262); Myc-tag (SEQ ID NO: 6); NE-tag(SEQ ID NO: 264); Rho1D4-tag (SEQ ID NO: 265); S-tag (SEQ ID NO: 266);SBP-tag (SEQ ID NO: 267); Softag-1 (SEQ ID NO: 268); Softag-2 (SEQ IDNO: 269); Spot-tag (SEQ ID NO: 270); Strep-tag (SEQ ID NO: 271); TC tag(SEQ ID NO: 272); Ty tag (SEQ ID NO: 273); V5 tag (SEQ ID NO: 3);VSV-tag (SEQ ID NO: 275); and Xpress tag (SEQ ID NO: 276).

363. The method of embodiment 360, wherein the peptide tag is selectedfrom the group consisting of: AU1 epitope (SEQ ID NO: 278); AU5 epitope(SEQ ID NO: 279); Bacteriophage T7 epitope (T7-tag) (SEQ ID NO: 280);Bluetongue virus tag (B-tag) (SEQ ID NO: 281); E2 epitope (SEQ ID NO:282); Histidine affinity tag (HAT) (SEQ ID NO: 283); HSV epitope (SEQ IDNO: 284); Polyarginine (Arg-tag) (SEQ ID NO: 285); Polyaspartate(Asp-tag) (SEQ ID NO: 286); Polyphenylalanine (Phe-tag) (SEQ ID NO:287); S1-tag (SEQ ID NO: 288); S-tag (SEQ ID NO: 266); and VSV-tag (SEQID NO: 275).

364. The method of embodiment 359, wherein the functional moiety is animmunoepitope.

365. The method of embodiment 364, wherein the immunoepitope is selectedfrom the group consisting of: tetanus toxoid (SEQ ID NO: 396);diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1(SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumpsimmunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406);hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1(SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes towardclass I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA(SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO:420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422);hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO:424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ IDNO: 426).

366. The method of embodiment 359, wherein the functional moiety altersthe localization of the protein of interest.

367. The method of embodiment 359, wherein the functional moiety is adegradation tag such that the degradation rate of the protein ofinterest is altered.

368. The method of embodiment 367, wherein the degradation tag resultsin the elimination of the tagged protein.

369. The method of embodiment 359, wherein the functional moiety is asmall molecule binding domain.

370. The method of embodiment 359, wherein the small molecule bindingdomain is FKBP12 of SEQ ID NO: 488.

371. The method of embodiment 359, wherein the small molecule bindingdomain is FKBP12-F36V of SEQ ID NO: 489.

372. The method of embodiment 359, wherein the small molecule bindingdomain is cyclophilin of SEQ ID NOs: 490 and 493-494.

373. The method of embodiment 359, wherein the small molecule bindingdomain is installed in two or more proteins of interest.

374. The method of embodiment 373, wherein the two or more proteins ofinterest may dimerize upon contacting with a small molecule.

375. The method of embodiment 369, wherein the small molecule is a dimerof a small molecule selected from the group consisting of:

376. A method of installing an immunoepitope in a protein of interestencoded by a target nucleotide sequence by prime editing, the methodcomprising: (a) contacting the target nucleotide sequence with a (i)prime editor comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edittemplate encoding the functional moiety; (b) polymerizing a singlestrand DNA sequence encoding the immunoepitope; and (c) incorporatingthe single strand DNA sequence in place of a corresponding endogenousstrand at the target nucleotide sequence through a DNA repair and/orreplication process, wherein the method produces a recombinant targetnucleotide sequence that encodes a fusion protein comprising the proteinof interest and the immunoepitope.

377. The method of embodiment 376, wherein the immunoepitope is selectedfrom the group consisting of: tetanus toxoid (SEQ ID NO: 396);diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1(SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumpsimmunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406);hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1(SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes towardclass I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA(SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO:420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422);hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO:424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ IDNO: 426).

378. The method of embodiment 376, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

379. The method of embodiment 376, wherein the napDNAbp is a Cas9nickase (nCas9).

380. The method of embodiment 376, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

381. The method of embodiment 376, wherein the PEgRNA comprises SEQ IDNOs: 101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761,776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540,3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929,3972-3989.

382. A method of installing a small molecule dimerization domain in aprotein of interest encoded by a target nucleotide sequence by primeediting, the method comprising: (a) contacting the target nucleotidesequence with a (i) prime editor comprising a nucleic acid programmableDNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNAcomprising an edit template encoding the small molecule dimerizationdomain; (b) polymerizing a single strand DNA sequence encoding theimmunoepitope; and (c) incorporating the single strand DNA sequence inplace of a corresponding endogenous strand at the target nucleotidesequence through a DNA repair and/or replication process, wherein themethod produces a recombinant target nucleotide sequence that encodes afusion protein comprising the protein of interest and the small moleculedimerization domain.

383. The method of embodiment 382, further comprising conducting themethod on a second protein of interest.

384. The method of embodiment 383, wherein the first protein of interestand the second protein of interest dimerize in the presence of a smallmolecule that binds to the dimerization domain on each of said proteins.

385. The method of embodiment 382, wherein the small molecule bindingdomain is FKBP12 of SEQ ID NO: 488.

386. The method of embodiment 382, wherein the small molecule bindingdomain is FKBP12-F36V of SEQ ID NO: 489.

387. The method of embodiment 382, wherein the small molecule bindingdomain is cyclophilin of SEQ ID NOs: 490 and 493-494.

388. The method of embodiment 382, wherein the small molecule is a dimerof a small molecule selected from the group consisting of:

389. The method of embodiment 382, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

390. The method of embodiment 382, wherein the napDNAbp is a Cas9nickase (nCas9).

391. The method of embodiment 382, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

392. The method of embodiment 382, wherein the PEgRNA comprises SEQ IDNOs: 101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761,776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540,3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929,3972-3989.

393. A method of installing a peptide tag or epitope onto a proteinusing prime editing, comprising: contacting a target nucleotide sequenceencoding the protein with a prime editor construct configured to inserttherein a second nucleotide sequence encoding the peptide tag to resultin a recombinant nucleotide sequence, such that the peptide tag and theprotein are expressed from the recombinant nucleotide sequence as afusion protein.

394. The method of embodiment 383, wherein the peptide tag is used forpurification and/or detection of the protein.

395. The method of embodiment 383, wherein the peptide tag is apoly-histidine (e.g., HHHHHH (SEQ ID NO: 252-262)), FLAG (e.g., DYKDDDDK(SEQ ID NO: 2)), V5 (e.g., GKPIPNPLLGLDST (SEQ ID NO: 3)), GCN4, HA(e.g., YPYDVPDYA (SEQ ID NO: 5)), Myc (e.g. EQKLISEED (SEQ ID NO: 6)),or GST.

396. The method of embodiment 383, wherein the peptide tag has an aminoacid sequence selected from the group consisting of SEQ ID NO: 245-290.

397. The method of embodiment 383, wherein the peptide tag is fused tothe protein by a linker.

398. The method of embodiment 383, wherein the fusion protein has thefollowing structure: [protein]-[peptide tag] or [peptide tag]-[protein],wherein “]-[” represents an optional linker.

399. The method of embodiment 383, wherein the linker has an amino acidsequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

400. The method of embodiment 383, wherein the prime editor constructcomprises a PEgRNA comprising the nucleotide sequence of SEQ ID NOs:18-101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777,2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556,3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, 3972-3989.

401. The method of embodiment 383, wherein the PEgRNA comprises aspacer, a gRNA core, and an extension arm, wherein the spacer iscomplementary to the target nucleotide sequence and the extension armcomprises a reverse transcriptase template that encodes the peptide tag.

402. The method of embodiment 383, wherein the PEgRNA comprises aspacer, a gRNA core, and an extension arm, wherein the spacer iscomplementary to the target nucleotide sequence and the extension armcomprises a reverse transcriptase template that encodes the peptide tag.

403. A method of preventing or halting the progression of a priondisease by installing on or more protective mutations into PRNP encodedby a target nucleotide sequence by prime editing, the method comprising:(a) contacting the target nucleotide sequence with a (i) prime editorcomprising a nucleic acid programmable DNA binding protein (napDNAbp)and a polymerase and (ii) a PEgRNA comprising an edit template encodingthe functional moiety; (b) polymerizing a single strand DNA sequenceencoding the protective mutation; and (c) incorporating the singlestrand DNA sequence in place of a corresponding endogenous strand at thetarget nucleotide sequence through a DNA repair and/or replicationprocess, wherein the method produces a recombinant target nucleotidesequence that encodes a PRNP comprising a protective mutation and whichis resistant to misfolding.

404. The method of embodiment 403, wherein the prion disease is a humanprion disease.

405. The method of embodiment 403, wherein the prion disease is ananimal prion disease.

406. The method of embodiment 404, wherein the prion disease isCreutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease(vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal FamilialInsomnia, or Kuru.

407. The method of embodiment 403, wherein the prion disease is BovineSpongiform Encephalopathy (BSE or “mad cow disease”), Chronic WastingDisease (CWD), Scrapie, Transmissible Mink Encephalopathy, FelineSpongiform Encephalopathy, and Ungulate Spongiform Encephalopathy.

408. The method of embodiment 403, wherein the wildtype PRNP amino acidsequence is SEQ ID NOs: 291-292.

409. The method of embodiment 403, wherein the method results in amodified PRNP amino acid sequence selected from the group consisting ofSEQ ID NOs: 293-323, wherein said modified PRNP protein is resistant tomisfolding.

410. The method of embodiment 403, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

411. The method of embodiment 403, wherein the napDNAbp is a Cas9nickase (nCas9).

412. The method of embodiment 403, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs:18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

413. The method of embodiment 403, wherein the PEgRNA comprises SEQ IDNOs: 101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761,776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540,3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929,3972-3989.

414. A method of installing a ribonucleotide motif or tag in an RNA ofinterest encoded by a target nucleotide sequence by prime editing, themethod comprising: (a) contacting the target nucleotide sequence with a(i) prime editor comprising a nucleic acid programmable DNA bindingprotein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edittemplate encoding the ribonucleotide motif or tag; (b) polymerizing asingle strand DNA sequence encoding the ribonucleotide motif or tag; and(c) incorporating the single strand DNA sequence in place of acorresponding endogenous strand at the target nucleotide sequencethrough a DNA repair and/or replication process, wherein the methodproduces a recombinant target nucleotide sequence that encodes amodified RNA of interest comprising the ribonucleotide motif or tag.

415. The method of embodiment 414, wherein ribonucleotide motif or tagis a detection moiety.

416. The method of embodiment 414, wherein the ribonucleotide motif ortag affects the expression level of the RNA of interest.

417. The method of embodiment 414, wherein the ribonucleotide motif ortag affects the transport or subcellular location of the RNA ofinterest.

418. The method of embodiment 414, wherein the ribonucleotide motif ortag is selected from the group consisting of SV40 type 1, SV40 type 2,SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE,Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer,spinach aptamer, mango aptamer, HDV ribozyme, and m6A.

419. The method of embodiment 414, wherein the PEgRNA comprises SEQ IDNOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

420. The method of embodiment 414, wherein the fusion protein comprisesthe amino acid sequence of PE1, PE2, or PE3.

421. The method of embodiment 414, wherein the napDNAbp is a Cas9nickase (nCas9).

422. The method of embodiment 414, wherein the napDNAbp comprises theamino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

423. A method of installing or deleting a functional moiety in a proteinof interest encoded by a target nucleotide sequence by prime editing,the method comprising: (a) contacting the target nucleotide sequencewith a (i) prime editor comprising a nucleic acid programmable DNAbinding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprisingan edit template encoding the functional moiety or deletion of same; (b)polymerizing a single strand DNA sequence encoding the functional moietyor deletion of same; and (c) incorporating the single strand DNAsequence in place of a corresponding endogenous strand at the targetnucleotide sequence through a DNA repair and/or replication process,wherein the method produces a recombinant target nucleotide sequencethat encodes a modified protein comprising the protein of interest andthe functional moiety or the removal of same, wherein the functionalmoiety alters a modification state or localization state of the protein.

424. The method of embodiment 423, wherein functional moiety alters thephosphorylation, ubiquitylation, glycosylation, lipidation,hydroxylation, methylation, acetylation, crotonylation, SUMOylationstate of the protein of interest.

Group A. Fusion Proteins, Guides, and Methods

Embodiment 1. A fusion protein comprising a nucleic acid programmableDNA binding protein (napDNAbp) and a reverse transcriptase.

Embodiment 2. The fusion protein of embodiment 1, wherein the fusionprotein is capable of carrying out genome editing by target-primedreverse transcription in the presence of an extended guide RNA.

Embodiment 3. The fusion protein of embodiment 1, wherein the napDNAbphas a nickase activity.

Embodiment 4. The fusion protein of embodiment 1, wherein the napDNAbpis a Cas9 protein or variant thereof.

Embodiment 5. The fusion protein of embodiment 1, wherein the napDNAbpis a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

Embodiment 6. The fusion protein of embodiment 1, wherein the napDNAbpis Cas9 nickase (nCas9).

Embodiment 7. The fusion protein of embodiment 1, wherein the napDNAbpis selected from the group consisting of: Cas9, CasX, CasY, Cpf1, C2c1,C2c2, C2C3, and Argonaute and optionally has a nickase activity.

Embodiment 8. The fusion protein of embodiment 1, wherein the fusionprotein when complexed with an extended guide RNA is capable of bindingto a target DNA sequence.

Embodiment 9. The fusion protein of embodiment 8, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand.

Embodiment 10. The fusion protein of embodiment 8, wherein the bindingof the fusion protein complexed to the extended guide RNA forms anR-loop.

Embodiment 11. The fusion protein of embodiment 10, wherein the R-loopcomprises (i) an RNA-DNA hybrid comprising the extended guide RNA andthe target strand, and (ii) the complementary non-target strand.

Embodiment 12. The fusion protein of embodiment 11, wherein thecomplementary non-target strand is nicked to form a reversetranscriptase priming sequence having a free 3′ end.

Embodiment 13. The fusion protein of embodiment 2, wherein the extendedguide RNA comprises (a) a guide RNA, and (b) an RNA extension at the 5′or the 3′ end of the guide RNA, or at an intramolecular location in theguide RNA.

Embodiment 14. The fusion protein of embodiment 13, wherein the RNAextension comprises (i) a reverse transcription template sequencecomprising a desired nucleotide change, (ii) a reverse transcriptionprimer binding site, and (iii) optionally, a linker sequence.

Embodiment 15. The fusion protein of embodiment 14, wherein the reversetranscription template sequence encodes a single-strand DNA flap that iscomplementary to an endogenous DNA sequence adjacent to the nick site,wherein the single-strand DNA flap comprises the desired nucleotidechange.

Embodiment 16. The fusion protein of embodiment 13, wherein the RNAextension is at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, atleast 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides,at least 19 nucleotides, at least 20 nucleotides, at least 21nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least24 nucleotides, or at least 25 nucleotides in length.

Embodiment 17. The fusion protein of embodiment 15, wherein thesingle-strand DNA flap hybridizes to the endogenous DNA sequenceadjacent to the nick site, thereby installing the desired nucleotidechange.

Embodiment 18. The fusion protein of embodiment 15, wherein thesingle-stranded DNA flap displaces the endogenous DNA sequence adjacentto the nick site and which has a free 5′ end.

Embodiment 19. The fusion protein of embodiment 18, wherein theendogenous DNA sequence having the 5′ end is excised by the cell.

Embodiment 20. The fusion protein of embodiment 18, wherein cellularrepair of the single-strand DNA flap results in installation of thedesired nucleotide change, thereby forming a desired product.

Embodiment 21. The fusion protein of embodiment 14, wherein the desirednucleotide change is installed in an editing window that is betweenabout −4 to +10 of the PAM sequence, or between about −10 to +20 of thePAM sequence, or between about −20 to +40 of the PAM sequence, orbetween about −30 to +100 of the PAM sequence, or wherein the desirednucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 nucleotides downstream of the nick site.

Embodiment 22. The fusion protein of embodiment 1, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18, or an amino acidsequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical tothe amino acid sequence to SEQ ID NO: 18.

Embodiment 23. The fusion protein of embodiment 1, wherein the napDNAbpcomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,98%, or 99% identical to the amino acid sequence of any one of SEQ IDNOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

The fusion protein of embodiment 1, wherein the reverse transcriptasecomprises any one of the amino acid sequences of SEQ ID NO: 89.

Embodiment 24. The fusion protein of embodiment 1, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

The fusion protein of embodiment 1, wherein the reverse transcriptasecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,98%, or 99% identical to the amino acid sequence of any one of SEQ IDNO: 89.

Embodiment 25. The fusion protein of embodiment 1, wherein the reversetranscriptase comprises an amino acid sequence that is at least 80%,85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of anyone of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154,159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 26. The fusion protein of embodiment 1, wherein the reversetranscriptase is a naturally-occurring reverse transcriptase from aretrovirus or a retrotransposon.

Embodiment 27. The fusion protein of any one of the previousembodiments, wherein the fusion protein comprises the structureNH₂-[napDNAbp]-[reverse transcriptase]-COOH; or NH₂-[reversetranscriptase]-[napDNAbp]-COOH, wherein each instance of “]-[” indicatesthe presence of an optional linker sequence.

Embodiment 28. The fusion protein of embodiment 27, wherein the linkersequence comprises an amino acid sequence of SEQ ID NOs: 127, 165-176,446, 453, and 767-769.

Embodiment 29. The fusion protein of embodiment 14, wherein the desirednucleotide change is a single nucleotide change, an insertion of one ormore nucleotides, or a deletion of one or more nucleotides.

Embodiment 30. The fusion protein of embodiment 29, wherein the insertor deletion is at least 1, at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33, at least 34, at least 35, at least 36, at least 37, at least 38, atleast 39, at least 40, at least 41, at least 42, at least 43, at least44, at least 45, at least 46, at least 47, at least 48, at least 49, orat least 50.

Embodiment 31. An extended guide RNA comprising a guide RNA and at leastone RNA extension.

Embodiment 32. The extended guide RNA of embodiment 1, wherein the RNAextension is position at the 3′ or 5′ end of the guide RNA, or at anintramolecular position in the guide RNA.

Embodiment 33. The extended guide RNA of embodiment 31, wherein theextended guide RNA is capable of binding to a napDNAbp and directing thenapDNAbp to a target DNA sequence.

Embodiment 34. The extended guide RNA of embodiment 33, wherein thetarget DNA sequence comprises a target strand and a complementarynon-target strand, wherein the guide RNA hybridizes to the target strandto form an RNA-DNA hybrid and an R-loop.

Embodiment 35. The extended guide RNA of embodiment 31, wherein the atleast one RNA extension comprises (i) a reverse transcription templatesequence, (ii) a reverse transcription primer binding site, and (iii)optionally a linker sequence.

Embodiment 36. The extended guide RNA of embodiment 35, wherein the RNAextension is at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, atleast 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides,at least 19 nucleotides, at least 20 nucleotides, at least 21nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least24 nucleotides, or at least 25 nucleotides in length.

Embodiment 37. The extended guide RNA of embodiment 35, wherein thereverse transcription template sequence is at least 3 nucleotides, atleast 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, atleast 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, atleast 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides,at least 13 nucleotides, at least 14 nucleotides, or at least 15nucleotides in length.

Embodiment 38. The extended guide RNA of embodiment 35, wherein thereverse transcription primer binding site sequence is at least 3nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or atleast 15 nucleotides in length.

Embodiment 39. The extended guide RNA of embodiment 35, wherein theoptional linker sequence is at least 3 nucleotides, at least 4nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides inlength.

Embodiment 40. The extended guide RNA of embodiment 35, wherein thereverse transcription template sequence encodes a single-strand DNA flapthat is complementary to an endogenous DNA sequence adjacent to a nicksite, wherein the single-strand DNA flap comprises a desired nucleotidechange.

Embodiment 41. The extended guide RNA of embodiment 40, wherein thesingle-stranded DNA flap displaces an endogenous single-strand DNAhaving a 5′ end in the target DNA sequence that has been nicked, andwherein the endogenous single-strand DNA is immediately adjacentdownstream of the nick site.

Embodiment 42. The extended guide RNA of embodiment 41, wherein theendogenous single-stranded DNA having the free 5′ end is excised by thecell.

Embodiment 43. The extended guide RNA of embodiment 41, wherein cellularrepair of the single-strand DNA flap results in installation of thedesired nucleotide change, thereby forming a desired product.

Embodiment 44. The extended guide RNA of embodiment 31, comprising thenucleotide sequence of SEQ ID NOs: 394, 429-442, 641-649, 678-692,2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and3755-3810, or a nucleotide sequence having at least 85%, or at least90%, or at least 95%, or at least 98%, or at least 99% sequence identitywith any one of SEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103,3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810.

Embodiment 45. The extended guide RNA of embodiment 35, wherein thereverse transcription template sequence comprises a nucleotide sequencethat is at least 80%, or 85%, or 90%, or 95%, or 99% identical to theendogenous DNA target.

Embodiment 46. The extended guide RNA of embodiment 35, wherein thereverse transcription primer binding site hybridizes with a free 3′ endof the cut DNA.

Embodiment 47. The extended guide RNA of embodiment 35, wherein theoptional linker sequence is at least 1 nucleotide, or at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15 nucleotides in length.

Embodiment 48. A complex comprising a fusion protein of any one ofembodiments 1-30 and an extended guide RNA.

Embodiment 49. The complex of embodiment 48, wherein the extended guideRNA comprises a guide RNA and an RNA extension at the 3′ or 5′ end ofthe guide RNA or at an intramolecular position in the guide RNA.

Embodiment 50. The complex of embodiment 48, wherein the extended guideRNA is capable of binding to a napDNAbp and directing the napDNAbp to atarget DNA sequence.

Embodiment 51. The complex of embodiment 50, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand, wherein the guide RNA hybridizes to the target strand to form anRNA-DNA hybrid and an R-loop.

Embodiment 52. The complex of embodiment 49, wherein the at least oneRNA extension comprises (i) a reverse transcription template sequence,(ii) a reverse transcription primer binding site, and (iii) optionally alinker sequence.

Embodiment 53. The complex of embodiment 48, wherein the extended guideRNA comprises the nucleotide sequence of SEQ ID NOs: 394, 429-442,641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556,3628-3698, and 3755-3810, or a nucleotide sequence having at least 85%,or at least 90%, or at least 95%, or at least 98%, or at least 99%sequence identity with any one of SEQ ID NOs: 394, 429-442, 641-649,678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556,3628-3698, and 3755-3810.

Embodiment 54. The complex of embodiment 52, wherein the reversetranscription template sequence comprises a nucleotide sequence havingat least 80%, or 85%, or 90%, or 95%, or 99% sequence identity with theendogenous DNA target.

Embodiment 55. The complex of embodiment 52, wherein the reversetranscription primer binding site hybridizes with a free 3′ end of thecut DNA.

Embodiment 56. A complex comprising a napDNAbp and an extended guideRNA.

Embodiment 57. The complex of embodiment 56, wherein the napDNAbp is aCas9 nickase.

Embodiment 58. The complex of embodiment 56, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18, or an amino acidsequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with SEQ ID NO: 18.

Embodiment 59. The complex of embodiment 57, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 60. The complex of embodiment 57, wherein the extended guideRNA comprises a guide RNA and an RNA extension at the 3′ or 5′ end ofthe guide RNA, or at an intramolecular position in the guide RNA.

Embodiment 61. The complex of embodiment 57, wherein the extended guideRNA is capable of directing the napDNAbp to a target DNA sequence.

Embodiment 62. The complex of embodiment 61, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand, wherein the spacer sequence hybridizes to the target strand toform an RNA-DNA hybrid and an R-loop.

Embodiment 63. The complex of embodiment 61, wherein the RNA extensioncomprises (i) a reverse transcription template sequence, (ii) a reversetranscription primer binding site, and (iii) optionally a linkersequence.

Embodiment 64. The complex of embodiment 57, wherein the extended guideRNA comprises the nucleotide sequence of SEQ ID NOs: 394, 429-442,641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556,3628-3698, and 3755-3810, or a nucleotide sequence having at least 85%,or at least 90%, or at least 95%, or at least 98%, or at least 99%sequence identity with any one of SEQ ID NOs: 394, 429-442, 641-649,678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556,3628-3698, and 3755-3810.

Embodiment 65. The complex of embodiment 63, wherein the reversetranscription template sequence comprises a nucleotide sequence that isat least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenousDNA target.

Embodiment 66. The complex of embodiment 63, wherein the reversetranscription primer binding site hybridizes with a free 3′ end of thecut DNA.

Embodiment 67. The complex of embodiment 63, wherein the optional linkersequence is at least 1 nucleotide, or at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, or at least 15nucleotides in length.

Embodiment 68. A polynucleotide encoding the fusion protein of any ofembodiments 1-30.

Embodiment 69. A vector comprising the polynucleotide of embodiment 68.

Embodiment 70. A cell comprising the fusion protein of any ofembodiments 1-30 and an extended guide RNA bound to the napDNAbp of thefusion protein.

Embodiment 71. A cell comprising a complex of any one of embodiments48-67.

Embodiment 72. A pharmaceutical composition comprising: (i) a fusionprotein of any of embodiments 1-30, the complex of embodiments 48-67,the polynucleotide of embodiment 68, or the vector of embodiment 69; and(ii) a pharmaceutically acceptable excipient.

Embodiment 73. A pharmaceutical composition comprising: (i) the complexof embodiments 48-67 (ii) reverse transcriptase provided in trans; and(iii) a pharmaceutically acceptable excipient.

Embodiment 74. A kit comprising a nucleic acid construct, comprising:(i) a nucleic acid sequencing encoding the fusion protein of any one ofembodiments 1-30; and (ii) a promoter that drives expression of thesequence of (i).

Embodiment 75. A method for installing a desired nucleotide change in adouble-stranded DNA sequence, the method comprising:

-   -   (i) contacting the double-stranded DNA sequence with a complex        comprising a fusion protein and an extended guide RNA, wherein        the fusion protein comprises a napDNAbp and a reverse        transcriptase and wherein the extended guide RNA comprises a        reverse transcription template sequence comprising the desired        nucleotide change;    -   (ii) nicking the double-stranded DNA sequence on the non-target        strand, thereby generating a free single-strand DNA having a 3′        end;    -   (iii) hybridizing the 3′ end of the free single-strand DNA to        the reverse transcription template sequence, thereby priming the        reverse transcriptase domain;    -   (iv) polymerizing a strand of DNA from the 3′ end, thereby        generating a single-strand DNA flap comprising the desired        nucleotide change;    -   (v) replacing an endogenous DNA strand adjacent the cut site        with the single-strand DNA flap, thereby installing the desired        nucleotide change in the double-stranded DNA sequence.

Embodiment 76. The method of embodiment 75, wherein the step of (v)replacing comprises: (i) hybridizing the single-strand DNA flap to theendogenous DNA strand adjacent the cut site to create a sequencemismatch; (ii) excising the endogenous DNA strand; and (iii) repairingthe mismatch to form the desired product comprising the desirednucleotide change in both strands of DNA.

Embodiment 77. The method of embodiment 76, wherein the desirednucleotide change is a single nucleotide substitution, a deletion, or aninsertion.

Embodiment 78. The method of embodiment 77, wherein the singlenucleotide substitution is a transition or a transversion.

Embodiment 79. The method of embodiment 76, wherein the desirednucleotide change is (1) a G to T substitution, (2) a G to Asubstitution, (3) a G to C substitution, (4) a T to G substitution, (5)a T to A substitution, (6) a T to C substitution, (7) a C to Gsubstitution, (8) a C to T substitution, (9) a C to A substitution, (10)an A to T substitution, (11) an A to G substitution, or (12) an A to Csubstitution.

Embodiment 80. The method of embodiment 76, wherein the desired nucleoidchange converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepairto an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:Abasepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) aT:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair,(8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:Tbasepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepairto a G:C basepair, or (12) an A:T basepair to a C:G basepair.

Embodiment 81. The method of embodiment 76, wherein the desirednucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25nucleotides.

Embodiment 82. The method of embodiment 76, wherein the desirednucleotide change corrects a disease-associated gene.

Embodiment 83. The method of embodiment 82, wherein thedisease-associated gene is associated with a monogenetic disorderselected from the group consisting of: Adenosine Deaminase (ADA)Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; DuchenneMuscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease;Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1;Pachyonychia Congenita; Phenylkeotnuria; Severe CombinedImmunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; andTay-Sachs Disease.

Embodiment 84. The method of embodiment 82, wherein thedisease-associated gene is associated with a polygenic disorder selectedfrom the group consisting of: heart disease; high blood pressure;Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 85. The method of embodiment 76, wherein the napDNAbp is anuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease activeCas9.

Embodiment 86. The method of embodiment 76, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18.

Embodiment 87. The method of embodiment 76, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 88. The method of embodiment 76, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 89. The method of embodiment 76, wherein the reversetranscriptase domain comprises an amino acid sequence having at least80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acidsequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139,143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and766.

Embodiment 90. The method of embodiment 76, wherein the extended guideRNA comprises an RNA extension at the 3′ or 5′ ends or at anintramolecular location in the guide RNA, wherein the RNA extensioncomprises the reverse transcription template sequence.

Embodiment 91. The method of embodiment 90, wherein the RNA extension isat least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides,at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides,at least 11 nucleotides, at least 12 nucleotides, at least 13nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, atleast 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides,at least 22 nucleotides, at least 23 nucleotides, at least 24nucleotides, or at least 25 nucleotides in length.

Embodiment 92. The method of embodiment 76, wherein the extended guideRNA has a nucleotide sequence selected from the group consisting of SEQID NOs: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455,3479-3493, 3522-3556, 3628-3698, and 3755-3810.

Embodiment 93. A method for introducing one or more changes in thenucleotide sequence of a DNA molecule at a target locus, comprising:

-   -   (i) contacting the DNA molecule with a nucleic acid programmable        DNA binding protein (napDNAbp) and a guide RNA which targets the        napDNAbp to the target locus, wherein the guide RNA comprises a        reverse transcriptase (RT) template sequence comprising at least        one desired nucleotide change;    -   (ii) forming an exposed 3′ end in a DNA strand at the target        locus;    -   (iii) hybridizing the exposed 3′ end to the RT template sequence        to prime reverse transcription;    -   (iv) synthesizing a single strand DNA flap comprising the at        least one desired nucleotide change based on the RT template        sequence by reverse transcriptase;    -   (v) and incorporating the at least one desired nucleotide change        into the corresponding endogenous DNA, thereby introducing one        or more changes in the nucleotide sequence of the DNA molecule        at the target locus.

Embodiment 94. The method of embodiment 93, wherein the one or morechanges in the nucleotide sequence comprises a transition.

Embodiment 95. The method of embodiment 94, wherein the transition isselected from the group consisting of: (a) T to C; (b) A to G; (c) C toT; and (d) G to A.

Embodiment 96. The method of embodiment 93, wherein the one or morechanges in the nucleotide sequence comprises a transversion.

Embodiment 97. The method of embodiment 96, wherein the transversion isselected from the group consisting of: (a) T to A; (b) T to G; (c) C toG; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.

Embodiment 98. The method of embodiment 93, wherein the one or morechanges in the nucleotide sequence comprises changing (1) a G:C basepairto a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:Cbasepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) aT:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair,(7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:Abasepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair toa T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:Tbasepair to a C:G basepair.

Embodiment 99. The method of embodiment 93, wherein the one or morechanges in the nucleotide sequence comprises an insertion or deletion of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25 nucleotides.

Embodiment 100. The method of embodiment 93, wherein the one or morechanges in the nucleotide sequence comprises a correction to adisease-associated gene.

Embodiment 101. The method of embodiment 100, wherein thedisease-associated gene is associated with a monogenetic disorderselected from the group consisting of: Adenosine Deaminase (ADA)Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; DuchenneMuscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease;Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1;Pachyonychia Congenita; Phenylkeotnuria; Severe CombinedImmunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; andTay-Sachs Disease.

Embodiment 102. The method of embodiment 100, wherein thedisease-associated gene is associated with a polygenic disorder selectedfrom the group consisting of: heart disease; high blood pressure;Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 103. The method of embodiment 93, wherein the napDNAbp is anuclease active Cas9 or variant thereof.

Embodiment 104. The method of embodiment 93, wherein the napDNAbp is anuclease inactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variantthereof.

Embodiment 105. The method of embodiment 93, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18.

Embodiment 106. The method of embodiment 93, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 107. The method of embodiment 93, wherein the reversetranscriptase is introduced in trans.

Embodiment 108. The method of embodiment 93, wherein the napDNAbpcomprises a fusion to a reverse transcriptase.

Embodiment 109. The method of embodiment 93, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 110. The method of embodiment 93, wherein the reversetranscriptase comprises an amino acid sequence having at least 80%, 85%,90%, 95%, 98%, or 99% sequence identity with the amino acid sequence ofany one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149,154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 111. The method of embodiment 93, wherein the step of formingan exposed 3′ end in the DNA strand at the target locus comprisesnicking the DNA strand with a nuclease.

Embodiment 112. The method of embodiment 111, wherein the nuclease isthe napDNAbp, is provided as a fusion domain of napDNAbp, or is providedin trans.

Embodiment 113. The method of embodiment 93, wherein the step of formingan exposed 3′ end in the DNA strand at the target locus comprisescontacting the DNA strand with a chemical agent.

Embodiment 114. The method of embodiment 93, wherein the step of formingan exposed 3′ end in the DNA strand at the target locus comprisesintroducing a replication error.

Embodiment 115. The method of embodiment 93, wherein the step ofcontacting the DNA molecule with the napDNAbp and the guide RNA forms anR-loop.

Embodiment 116. The method of embodiment 115, wherein the DNA strand inwhich the exposed 3′ end is formed is in the R-loop.

Embodiment 117. The method of embodiment 93, wherein guide RNA comprisesan extended portion that comprises the reverse transcriptase (RT)template sequence.

Embodiment 118. The method of embodiment 117, wherein the extendedportion is at the 3′ end of the guide RNA, the 5′ end of the guide RNA,or at an intramolecular position in the guide RNA.

Embodiment 119. The method of embodiment 93, wherein the guide RNAfurther comprises a primer binding site.

Embodiment 120. The method of embodiment 93, wherein the guide RNAfurther comprises a spacer sequence.

Embodiment 121. The method of embodiment 93, wherein the RT templatesequence is homologous to the corresponding endogenous DNA.

Embodiment 122. A method for introducing one or more changes in thenucleotide sequence of a DNA molecule at a target locus by target-primedreverse transcription, the method comprising: (a) contacting the DNAmolecule at the target locus with a (i) fusion protein comprising anucleic acid programmable DNA binding protein (napDNAbp) and a reversetranscriptase and (ii) a guide RNA comprising an RT template comprisinga desired nucleotide change; (b) conducting target-primed reversetranscription of the RT template to generate a single strand DNAcomprising the desired nucleotide change; and (c) incorporating thedesired nucleotide change into the DNA molecule at the target locusthrough a DNA repair and/or replication process.

Embodiment 123. The method of embodiment 122, wherein the RT template islocated at the 3′ end of the guide RNA, the 5′ end of the guide RNA, orat an intramolecular location in the guide RNA.

Embodiment 124. The method of embodiment 122, wherein the desirednucleotide change comprises a transition, a transversion, an insertion,or a deletion, or any combination thereof.

Embodiment 125. The method of claim 122, wherein the desired nucleotidechange comprises a transition selected from the group consisting of: (a)T to C; (b) A to G; (c) C to T; and (d) G to A.

Embodiment 126. The method of claim 122, wherein the desired nucleotidechange comprises a transversion selected from the group consisting of:(a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C;(g) G to C; and (h) G to T.

Embodiment 127. The method of embodiment 122, wherein the desirednucleotide change comprises changing (1) a G:C basepair to a T:Abasepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair toC:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepairto an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:Gbasepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) aC:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair,(11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:Gbasepair.

Embodiment 128. A polynucleotide encoding the extended guide RNA of anyone of embodiments 31-47.

Embodiment 129. A vector comprising the polynucleotide of embodiment128.

Embodiment 130. A cell comprising the vector of embodiment 129.

Embodiment 131. The fusion protein of any of embodiments 1-30, whereinthe reverse transcriptase is an error-prone reverse transcriptase.

Embodiment 132. A method for mutagenizing a DNA molecule at a targetlocus by target-primed reverse transcription, the method comprising: (a)contacting the DNA molecule at the target locus with a (i) fusionprotein comprising a nucleic acid programmable DNA binding protein(napDNAbp) and an error-prone reverse transcriptase and (ii) a guide RNAcomprising an RT template comprising a desired nucleotide change; (b)conducting target-primed reverse transcription of the RT template togenerate a mutagenized single strand DNA; and (c) incorporating themutagenized single strand DNA into the DNA molecule at the target locusthrough a DNA repair and/or replication process.

Embodiment 133. The method of embodiment 132, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 134. The method of embodiment 132, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 135. The method of embodiment 132, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 136. The method of embodiment 132, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 137. The method of embodiment 132, wherein the step of (b)conducting target-primed reverse transcription comprises generating a 3′end primer binding sequence at the target locus that is capable ofpriming reverse transcription by annealing to a primer binding site onthe guide RNA.

Embodiment 138. A method for replacing a trinucleotide repeat expansionmutation in a target DNA molecule with a healthy sequence comprising ahealthy number of repeat trinucleotides, the method comprising: (a)contacting the DNA molecule at the target locus with a (i) fusionprotein comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprisingan RT template comprising the replacement sequence, wherein said fusionprotein intr; (b) conducting target-primed reverse transcription of theRT template to generate a single strand DNA comprising the replacementsequence; and (c) incorporating the single strand DNA into the DNAmolecule at the target locus through a DNA repair and/or replicationprocess.

Embodiment 139. The method of embodiment 138, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 140. The method of embodiment 138, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 141. The method of embodiment 138, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 142. The method of embodiment 138, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 143. The method of embodiment 138, wherein the step of (b)conducting target-primed reverse transcription comprises generating a 3′end primer binding sequence at the target locus that is capable ofpriming reverse transcription by annealing to a primer binding site onthe guide RNA.

Embodiment 144. The method of embodiment 138, wherein the trinucleotiderepeat expansion mutation is associated with Huntington's Disease,Fragile X syndrome, or Friedreich's ataxia.

Embodiment 145. The method of embodiment 138, wherein the trinucleotiderepeat expansion mutation comprises a repeating unit of CAG triplets.

Embodiment 146. The method of embodiment 138, wherein the trinucleotiderepeat expansion mutation comprises a repeating unit of GAA triplets.

Embodiment 147. A method of installing a functional moiety in a proteinof interest encoded by a target nucleotide sequence by prime editing,the method comprising: (a) contacting the target nucleotide sequencewith a (i) prime editor comprising a nucleic acid programmable DNAbinding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNAcomprising an edit template encoding the functional moiety; (b)polymerizing a single strand DNA sequence encoding the functionalmoiety; and (c) incorporating the single strand DNA sequence in place ofa corresponding endogenous strand at the target nucleotide sequencethrough a DNA repair and/or replication process, wherein the methodproduces a recombinant target nucleotide sequence that encodes a fusionprotein comprising the protein of interest and the functional moiety.

Embodiment 148. The method of embodiment 147, wherein functional moietyis peptide tag.

Embodiment 149. The method of embodiment 148, wherein the peptide tag isan affinity tag, solubilization tag, chromatography tag, epitope tag, ora fluorescence tag.

Embodiment 150. The method of embodiment 148, wherein the peptide tag isselected from the group consisting of: AviTag (SEQ ID NO: 245); C-tag(SEQ ID NO: 246); Calmodulin-tag (SEQ ID NO: 247); polyglutamate tag(SEQ ID NO: 248); E-tag (SEQ ID NO: 249); FLAG-tag (SEQ ID NO: 2);HA-tag (SEQ ID NO: 5); His-tag (SEQ ID NOs: 252-262); Myc-tag (SEQ IDNO: 6); NE-tag (SEQ ID NO: 264); Rho1D4-tag (SEQ ID NO: 265); S-tag (SEQID NO: 266); SBP-tag (SEQ ID NO: 267); Softag-1 (SEQ ID NO: 268);Softag-2 (SEQ ID NO: 269); Spot-tag (SEQ ID NO: 270); Strep-tag (SEQ IDNO: 271); TC tag (SEQ ID NO: 272); Ty tag (SEQ ID NO: 273); V5 tag (SEQID NO: 3); VSV-tag (SEQ ID NO: 275); and Xpress tag (SEQ ID NO: 276).

Embodiment 151. The method of embodiment 148, wherein the peptide tag isselected from the group consisting of: AU1 epitope (SEQ ID NO: 278); AU5epitope (SEQ ID NO: 279); Bacteriophage T7 epitope (T7-tag) (SEQ ID NO:280); Bluetongue virus tag (B-tag) (SEQ ID NO: 281); E2 epitope (SEQ IDNO: 282); Histidine affinity tag (HAT) (SEQ ID NO: 283); HSV epitope(SEQ ID NO: 284); Polyarginine (Arg-tag) (SEQ ID NO: 285); Polyaspartate(Asp-tag) (SEQ ID NO: 286); Polyphenylalanine (Phe-tag) (SEQ ID NO:287); S1-tag (SEQ ID NO: 288); S-tag (SEQ ID NO: 266); and VSV-G (SEQ IDNO: 275).

Embodiment 152. The method of embodiment 147, wherein the functionalmoiety is an immunoepitope.

Embodiment 153. The method of embodiment 152, wherein the immunoepitopeis selected from the group consisting of: tetanus toxoid (SEQ ID NO:396); diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumpsimmunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO:402); mumps immunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO:406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410);TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopestoward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward classI HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO:422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ IDNO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQID NO: 426).

Embodiment 154. The method of embodiment 147, wherein the functionalmoiety alters the localization of the protein of interest.

Embodiment 155. The method of embodiment 147, wherein the functionalmoiety is a degradation tag such that the degradation rate of theprotein of interest is altered.

Embodiment 156. The method of embodiment 155, wherein the degradationtag comprises an amino acid sequence encoding the degradation tags asdisclosed herein.

Embodiment 157. The method of embodiment 147, wherein the functionalmoiety is a small molecule binding domain.

Embodiment 158. The method of embodiment 157, wherein the small moleculebinding domain is FKBP12 of SEQ ID NO: 488.

Embodiment 159. The method of embodiment 157, wherein the small moleculebinding domain is FKBP12-F36V of SEQ ID NO: 489.

Embodiment 160. The method of embodiment 157, wherein the small moleculebinding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.

Embodiment 161. The method of embodiment 157, wherein the small moleculebinding domain is installed in two or more proteins of interest.

Embodiment 162. The method of embodiment 161, wherein the two or moreproteins of interest may dimerize upon contacting with a small molecule.

Embodiment 163. The method of embodiment 157, wherein the small moleculeis a dimer of a small molecule selected from the group consisting ofthose compounds disclosed in Embodiment 163 of Group 1.

Embodiment 164. A method of installing an immunoepitope in a protein ofinterest encoded by a target nucleotide sequence by prime editing, themethod comprising: (a) contacting the target nucleotide sequence with a(i) prime editor comprising a nucleic acid programmable DNA bindingprotein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNAcomprising an edit template encoding the functional moiety; (b)polymerizing a single strand DNA sequence encoding the immunoepitope;and (c) incorporating the single strand DNA sequence in place of acorresponding endogenous strand at the target nucleotide sequencethrough a DNA repair and/or replication process, wherein the methodproduces a recombinant target nucleotide sequence that encodes a fusionprotein comprising the protein of interest and the immunoepitope.

Embodiment 165. The method of embodiment 164, wherein the immunoepitopeis selected from the group consisting of: tetanus toxoid (SEQ ID NO:396); diphtheria toxin mutant CRM197 (SEQ ID NO: 630); mumpsimmunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO:402); mumps immunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO:406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410);TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopestoward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward classI HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO:422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ IDNO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQID NO: 426).

Embodiment 166. The method of embodiment 164, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 167. The method of embodiment 164, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 168. The method of embodiment 164, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 169. The method of embodiment 164, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 170. A method of installing a small molecule dimerizationdomain in a protein of interest encoded by a target nucleotide sequenceby prime editing, the method comprising: (a) contacting the targetnucleotide sequence with a (i) prime editor comprising a nucleic acidprogrammable DNA binding protein (napDNAbp) and a reverse transcriptaseand (ii) a PEgRNA comprising an edit template encoding the smallmolecule dimerization domain; (b) polymerizing a single strand DNAsequence encoding the immunoepitope; and (c) incorporating the singlestrand DNA sequence in place of a corresponding endogenous strand at thetarget nucleotide sequence through a DNA repair and/or replicationprocess, wherein the method produces a recombinant target nucleotidesequence that encodes a fusion protein comprising the protein ofinterest and the small molecule dimerization domain.

Embodiment 171. The method of embodiment 170, further comprisingconducting the method on a second protein of interest.

Embodiment 172. The method of embodiment 171, wherein the first proteinof interest and the second protein of interest dimerize in the presenceof a small molecule that binds to the dimerization domain on each ofsaid proteins.

Embodiment 173. The method of embodiment 170, wherein the small moleculebinding domain is FKBP12 of SEQ ID NO: 488.

Embodiment 174. The method of embodiment 170, wherein the small moleculebinding domain is FKBP12-F36V of SEQ ID NO: 489.

Embodiment 175. The method of embodiment 170, wherein the small moleculebinding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.

Embodiment 176. The method of embodiment 170, wherein the small moleculeis a dimer of a small molecule selected from the group consisting ofthose compounds disclosed in Embodiment 163 of Group 1.

Embodiment 177. The method of embodiment 170, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 178. The method of embodiment 170, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 179. The method of embodiment 170, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 180. The method of embodiment 170, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 181. A method of installing a peptide tag or epitope onto aprotein using prime editing, comprising: contacting a target nucleotidesequence encoding the protein with a prime editor construct configuredto insert therein a second nucleotide sequence encoding the peptide tagto result in a recombinant nucleotide sequence, such that the peptidetag and the protein are expressed from the recombinant nucleotidesequence as a fusion protein.

Embodiment 182. The method of embodiment 181, wherein the peptide tag isused for purification and/or detection of the protein.

Embodiment 183. The method of embodiment 181, wherein the peptide tag isa poly-histidine (e.g., HHHHHH) (SEQ ID NOs: 252-262), FLAG (e.g.,DYKDDDDK) (SEQ ID NO: 2), V5 (e.g., GKPIPNPLLGLDST) (SEQ ID NO: 3),GCN4, HA (e.g., YPYDVPDYA) (SEQ ID NO: 5), Myc (e.g. EQKLISEED) (SEQ IDNO: 6), GST . . . etc.

Embodiment 184. The method of embodiment 181, wherein the peptide taghas an amino acid sequence selected from the group consisting of SEQ IDNO: 1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622.

Embodiment 185. The method of embodiment 181, wherein the peptide tag isfused to the protein by a linker.

Embodiment 186. The method of embodiment 181, wherein the fusion proteinhas the following structure: [protein]-[peptide tag] or [peptidetag]-[protein], wherein “]-[” represents an optional linker.

Embodiment 187. The method of embodiment 181, wherein the linker has anamino acid sequence of SEQ ID NO: 127, 165-176, 446,453, and 767-769.

Embodiment 188. The method of embodiment 181, wherein the prime editorconstruct comprises a PEgRNA comprising the nucleotide sequence of SEQID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 189. The method of embodiment 181, wherein the PEgRNAcomprises a spacer, a gRNA core, and an extension arm, wherein thespacer is complementary to the target nucleotide sequence and theextension arm comprises a reverse transcriptase template that encodesthe peptide tag.

Embodiment 190. The method of embodiment 181, wherein the PEgRNAcomprises a spacer, a gRNA core, and an extension arm, wherein thespacer is complementary to the target nucleotide sequence and theextension arm comprises a reverse transcriptase template that encodesthe peptide tag.

Embodiment 191. A method of preventing or halting the progression of aprion disease by installing on or more protective mutations into PRNPencoded by a target nucleotide sequence by prime editing, the methodcomprising: (a) contacting the target nucleotide sequence with a (i)prime editor comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising anedit template encoding the functional moiety; (b) polymerizing a singlestrand DNA sequence encoding the protective mutation; and (c)incorporating the single strand DNA sequence in place of a correspondingendogenous strand at the target nucleotide sequence through a DNA repairand/or replication process, wherein the method produces a recombinanttarget nucleotide sequence that encodes a PRNP comprising a protectivemutation and which is resistant to misfolding.

Embodiment 192. The method of embodiment 191, wherein the prion diseaseis a human prion disease.

Embodiment 193. The method of embodiment 191, wherein the prion diseaseis an animal prion disease.

Embodiment 194. The method of embodiment 192, wherein the prion diseaseis Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease(vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal FamilialInsomnia, or Kuru.

Embodiment 195. The method of embodiment 193, wherein the prion diseaseis Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), ChronicWasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy,Feline Spongiform Encephalopathy, and Ungulate SpongiformEncephalopathy.

Embodiment 196. The method of embodiment 191, wherein the wildtype PRNPamino acid sequence is SEQ ID NOs: 291-292.

Embodiment 197. The method of embodiment 191, wherein the method resultsin a modified PRNP amino acid sequence selected from the groupconsisting of SEQ ID NOs: 293-309, and 311-323, wherein said modifiedPRNP protein is resistant to misfolding.

Embodiment 198. The method of embodiment 191, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 199. The method of embodiment 191, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 200. The method of embodiment 191, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 201. The method of embodiment 191, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 202. A method of installing a ribonucleotide motif or tag inan RNA of interest encoded by a target nucleotide sequence by primeediting, the method comprising: (a) contacting the target nucleotidesequence with a (i) prime editor comprising a nucleic acid programmableDNA binding protein (napDNAbp) and a reverse transcriptase and (ii) aPEgRNA comprising an edit template encoding the ribonucleotide motif ortag; (b) polymerizing a single strand DNA sequence encoding theribonucleotide motif or tag; and (c) incorporating the single strand DNAsequence in place of a corresponding endogenous strand at the targetnucleotide sequence through a DNA repair and/or replication process,wherein the method produces a recombinant target nucleotide sequencethat encodes a modified RNA of interest comprising the ribonucleotidemotif or tag.

Embodiment 203. The method of embodiment 202, wherein ribonucleotidemotif or tag is a detection moiety.

Embodiment 204. The method of embodiment 202, wherein the ribonucleotidemotif or tag affects the expression level of the RNA of interest.

Embodiment 205. The method of embodiment 202, wherein the ribonucleotidemotif or tag affects the transport or subcellular location of the RNA ofinterest.

Embodiment 206. The method of embodiment 202, wherein the ribonucleotidemotif or tag is selected from the group consisting of SV40 type 1, SV40type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PANENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer,spinach aptamer, mango aptamer, HDV ribozyme, and m6A.

Embodiment 207. The method of embodiment 202, wherein the PEgRNAcomprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 499-505, 735-761, 776-777.

Embodiment 208. The method of embodiment 202, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 209. The method of embodiment 202, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 210. The method of embodiment 202, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 211. A method of installing or deleting a functional moietyin a protein of interest encoded by a target nucleotide sequence byprime editing, the method comprising: (a) contacting the targetnucleotide sequence with a (i) prime editor comprising a nucleic acidprogrammable DNA binding protein (napDNAbp) and a reverse transcriptaseand (ii) a PEgRNA comprising an edit template encoding the functionalmoiety or deletion of same; (b) polymerizing a single strand DNAsequence encoding the functional moiety or deletion of same; and (c)incorporating the single strand DNA sequence in place of a correspondingendogenous strand at the target nucleotide sequence through a DNA repairand/or replication process, wherein the method produces a recombinanttarget nucleotide sequence that encodes a modified protein comprisingthe protein of interest and the functional moiety or the removal ofsame, wherein the functional moiety alters a modification state orlocalization state of the protein.

Embodiment 212. The method of embodiment 211, wherein functional moietyalters the phosphorylation, ubiquitylation, glycosylation, lipidation,hydroxylation, methylation, acetylation, crotonylation, SUMOylationstate of the protein of interest.

Embodiment 213. A fusion protein comprising a nucleic acid programmableDNA binding protein (napDNAbp) and a polymerase.

Embodiment 214. The fusion protein of embodiment 213, wherein the fusionprotein is capable of carrying out prime editing in the presence of anprime editing guide RNA (PEgRNA).

Embodiment 215. The fusion protein of embodiment 213, wherein thenapDNAbp has a nickase activity.

Embodiment 216. The fusion protein of embodiment 213, wherein thenapDNAbp is a Cas9 protein or variant thereof.

Embodiment 217. The fusion protein of embodiment 213, wherein thenapDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), ora Cas9 nickase (nCas9).

Embodiment 218. The fusion protein of embodiment 213, wherein thenapDNAbp is Cas9 nickase (nCas9).

Embodiment 219. The fusion protein of embodiment 213, wherein thenapDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d,Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has anickase activity.

Embodiment 220. The fusion protein of embodiment 213, wherein the fusionprotein when complexed with a PEgRNA is capable of binding to a targetDNA sequence.

Embodiment 221. The fusion protein of embodiment 220, wherein the targetDNA sequence comprises a target strand and a complementary non-targetstrand.

Embodiment 222. The fusion protein of embodiment 220, wherein thebinding of the fusion protein complexed to the PEgRNA forms an R-loop.

Embodiment 223. The fusion protein of embodiment 222, wherein the R-loopcomprises (i) an RNA-DNA hybrid comprising the PEgRNA and the targetstrand, and (ii) the complementary non-target strand.

Embodiment 224. The fusion protein of embodiment 223, wherein thecomplementary non-target strand is nicked to form a priming sequencehaving a free 3′ end.

Embodiment 225. The fusion protein of embodiment 214, wherein the PEgRNAcomprises (a) a guide RNA and (b) an extension arm at the 5′ or the 3′end of the guide RNA, or at an intramolecular location in the guide RNA.

Embodiment 226. The fusion protein of embodiment 225, wherein theextension arm comprises (i) a DNA synthesis template sequence comprisinga desired nucleotide change, and (ii) a primer binding site.

Embodiment 227. The fusion protein of embodiment 226, wherein the DNAsynthesis template sequence encodes a single-strand DNA flap that iscomplementary to an endogenous DNA sequence adjacent to the nick site,wherein the single-strand DNA flap comprises the desired nucleotidechange.

Embodiment 228. The fusion protein of embodiment 225, wherein theextension arm is at least 5 nucleotides, at least 6 nucleotides, atleast 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, atleast 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides,at least 13 nucleotides, at least 14 nucleotides, at least 15nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, atleast 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides,at least 24 nucleotides, or at least 25 nucleotides in length.

Embodiment 229. The fusion protein of embodiment 227, wherein thesingle-strand DNA flap hybridizes to the endogenous DNA sequenceadjacent to the nick site, thereby installing the desired nucleotidechange.

Embodiment 230. The fusion protein of embodiment 227, wherein thesingle-stranded DNA flap displaces the endogenous DNA sequence adjacentto the nick site and which has a free 5′ end.

Embodiment 231. The fusion protein of embodiment 230, wherein theendogenous DNA sequence having the 5′ end is excised by the cell.

Embodiment 232. The fusion protein of embodiment 230, wherein cellularrepair of the single-strand DNA flap results in installation of thedesired nucleotide change, thereby forming a desired product.

Embodiment 233. The fusion protein of embodiment 226, wherein thedesired nucleotide change is installed in an editing window that isbetween about −4 to +10 of the PAM sequence, or between about −10 to +20of the PAM sequence, or between about −20 to +40 of the PAM sequence, orbetween about −30 to +100 of the PAM sequence, or wherein the desirednucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 nucleotides downstream of the nick site.

Embodiment 234. The fusion protein of embodiment 213, wherein thenapDNAbp comprises an amino acid sequence of SEQ ID NO: 18, or an aminoacid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with the amino acid sequence to SEQ ID NO: 18.

Embodiment 235. The fusion protein of embodiment 213, wherein thenapDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%,95%, 98%, or 99% sequence identity with the amino acid sequence of anyone of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460,467, and 482-487.

Embodiment 236. The fusion protein of embodiment 213, wherein thepolymerase is a reverse transcriptase comprising any one of the aminoacid sequences of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143,149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 237. The fusion protein of embodiment 213, wherein thepolymerase is a reverse transcriptase comprising an amino acid sequencehaving at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity withthe amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122,128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700,701-716, 739-741, and 766.

Embodiment 238. The fusion protein of embodiment 213, wherein thepolymerase is a naturally-occurring reverse transcriptase from aretrovirus or a retrotransposon.

Embodiment 239. The fusion protein of any one of the previousembodiments, wherein the fusion protein comprises the structureNH₂-[napDNAbp]-[polymerase]-COOH; or NH₂-[polymerase]-[napDNAbp]-COOH,wherein each instance of “]-[” indicates the presence of an optionallinker sequence.

Embodiment 240. The fusion protein of embodiment 239, wherein the linkersequence comprises an amino acid sequence of SEQ ID NOs: 127, 165-176,446, 453, and 767-769.

Embodiment 241. The fusion protein of embodiment 226, wherein thedesired nucleotide change is a single nucleotide change, an insertion ofone or more nucleotides, or a deletion of one or more nucleotides.

Embodiment 242. The fusion protein of embodiment 241, wherein the insertor deletion is at least 1, at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33, at least 34, at least 35, at least 36, at least 37, at least 38, atleast 39, at least 40, at least 41, at least 42, at least 43, at least44, at least 45, at least 46, at least 47, at least 48, at least 49, orat least 50.

Embodiment 243. A PEgRNA comprising a guide RNA and at least one nucleicacid extension arm comprising a DNA synthesis template.

Embodiment 244. The PEgRNA of embodiment 241, wherein the nucleic acidextension arm is position at the 3′ or 5′ end of the guide RNA, or at anintramolecular position in the guide RNA, and wherein the nucleic acidextension arm is DNA or RNA.

Embodiment 245. The PEgRNA of embodiment 242, wherein the PEgRNA iscapable of binding to a napDNAbp and directing the napDNAbp to a targetDNA sequence.

Embodiment 246. The PEgRNA of embodiment 245, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand, wherein the guide RNA hybridizes to the target strand to form anRNA-DNA hybrid and an R-loop.

Embodiment 247. The PEgRNA of embodiment 243, wherein the at least onenucleic acid extension arm comprises (i) a DNA synthesis template, and(ii) a primer binding site.

Embodiment 248. The PEgRNA of embodiment 247, wherein the nucleic acidextension arm is at least 5 nucleotides, at least 6 nucleotides, atleast 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, atleast 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides,at least 13 nucleotides, at least 14 nucleotides, at least 15nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, atleast 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides,at least 24 nucleotides, or at least 25 nucleotides in length.

Embodiment 249. The PEgRNA of embodiment 247, wherein the DNA synthesistemplate is at least 3 nucleotides, at least 4 nucleotides, at least 5nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, or at least 15 nucleotides in length.

Embodiment 250. The PEgRNA of embodiment 247, wherein the primer bindingsite is at least 3 nucleotides, at least 4 nucleotides, at least 5nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, or at least 15 nucleotides in length.

Embodiment 251. The PEgRNA of embodiment 243, further comprising atleast one additional structure selected from the group consisting of alinker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-proteinrecruitment domain.

Embodiment 252. The PEgRNA of embodiment 247, wherein the DNA synthesistemplate encodes a single-strand DNA flap that is complementary to anendogenous DNA sequence adjacent to a nick site, wherein thesingle-strand DNA flap comprises a desired nucleotide change.

Embodiment 253. The PEgRNA of embodiment 252, wherein thesingle-stranded DNA flap displaces an endogenous single-strand DNAhaving a 5′ end in the target DNA sequence that has been nicked, andwherein the endogenous single-strand DNA is immediately adjacentdownstream of the nick site.

Embodiment 254. The PEgRNA of embodiment 253, wherein the endogenoussingle-stranded DNA having the free 5′ end is excised by the cell.

Embodiment 255. The PEgRNA of embodiment 253, wherein cellular repair ofthe single-strand DNA flap results in installation of the desirednucleotide change, thereby forming a desired product.

Embodiment 256. The PEgRNA of embodiment 243, comprising the nucleotidesequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 499-505, 735-761, 776-777, or a nucleotide sequence having atleast 85%, or at least 90%, or at least 95%, or at least 98%, or atleast 99% sequence identity with any one of SEQ ID NOs: 101-104,181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.

Embodiment 257. The PEgRNA of embodiment 247, wherein the DNA synthesistemplate comprises a nucleotide sequence that is at least 80%, or 85%,or 90%, or 95%, or 99% identical to the endogenous DNA target.

Embodiment 258. The PEgRNA of embodiment 247, wherein the primer bindingsite hybridizes with a free 3′ end of the cut DNA.

Embodiment 259. The PEgRNA of embodiment 251, wherein the at least oneadditional structure is located at the 3′ or 5′ end of the PEgRNA.

Embodiment 260. A complex comprising a fusion protein of any one ofembodiments 213-242 and an PEgRNA.

Embodiment 261. The complex of embodiment 260, wherein the PEgRNAcomprises a guide RNA and an nucleic acid extension arm at the 3′ or 5′end of the guide RNA or at an intramolecular position in the guide RNA.

Embodiment 262. The complex of embodiment 260, wherein the PEgRNA iscapable of binding to a napDNAbp and directing the napDNAbp to a targetDNA sequence.

Embodiment 263. The complex of embodiment 262, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand, wherein the guide RNA hybridizes to the target strand to form anRNA-DNA hybrid and an R-loop.

Embodiment 264. The complex of embodiment 261, wherein the at least onenucleic acid extension arm comprises (i) a DNA synthesis template, and(ii) a primer binding site.

Embodiment 265. The complex of embodiment 260, wherein the PEgRNAcomprises the nucleotide sequence of SEQ ID NOs: 101-104, 181-183,223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or anucleotide sequence having at least 85%, or at least 90%, or at least95%, or at least 98%, or at least 99% sequence identity with any one ofSEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 266. The complex of embodiment 264, wherein the DNA synthesistemplate comprises a nucleotide sequence that is at least 80%, or 85%,or 90%, or 95%, or 99% identical to the endogenous DNA target.

Embodiment 267. The complex of embodiment 264, wherein the primerbinding site hybridizes with a free 3′ end of the cut DNA.

Embodiment 268. A complex comprising a napDNAbp and an PEgRNA.

Embodiment 269. The complex of embodiment 268, wherein the napDNAbp is aCas9 nickase.

Embodiment 270. The complex of embodiment 268, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18.

Embodiment 271. The complex of embodiment 268, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 272. The complex of embodiment 268, wherein the PEgRNAcomprises a guide RNA and a nucleic acid extension arm at the 3′ or 5′end of the guide RNA, or at an intramolecular position in the guide RNA.

Embodiment 273. The complex of embodiment 268, wherein the PEgRNA iscapable of directing the napDNAbp to a target DNA sequence.

Embodiment 274. The complex of embodiment 272, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand, wherein the spacer sequence of the PEgRNA hybridizes to thetarget strand to form an RNA-DNA hybrid and an R-loop.

Embodiment 275. The complex of embodiment 273, wherein the nucleic acidextension arm comprises (i) a DNA synthesis template, and (ii) a primerbinding site.

Embodiment 276. The complex of embodiment 269, wherein the PEgRNAcomprises the nucleotide sequence of SEQ ID NOs: 101-104, 181-183,223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or anucleotide sequence having at least 85%, or at least 90%, or at least95%, or at least 98%, or at least 99% sequence identity with any one ofSEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 277. The complex of embodiment 276, wherein the DNA synthesistemplate comprises a nucleotide sequence that is at least 80%, or 85%,or 90%, or 95%, or 99% identical to the endogenous DNA target.

Embodiment 278. The complex of embodiment 276, wherein the primerbinding site hybridizes with a free 3′ end of the cut DNA.

Embodiment 279. The complex of embodiment 276, wherein the PEgRNAfurther comprises at least one additional structure selected from thegroup consisting of a linker, a stem loop, a hairpin, a toeloop, anaptamer, or an RNA-protein recruitment domain.

Embodiment 280. A polynucleotide encoding the fusion protein of any ofembodiments 213-242.

Embodiment 281. A vector comprising the polynucleotide of embodiment280.

Embodiment 282. A cell comprising the fusion protein of any ofembodiments 213-242 and an PEgRNA bound to the napDNAbp of the fusionprotein.

Embodiment 283. A cell comprising a complex of any one of embodiments260-279.

Embodiment 284. A pharmaceutical composition comprising: (i) a fusionprotein of any of embodiments 213-242, the complex of embodiments260-279, the polynucleotide of embodiment 68, or the vector ofembodiment 69; and (ii) a pharmaceutically acceptable excipient.

Embodiment 285. A pharmaceutical composition comprising: (i) the complexof embodiments 260-279 (ii) a polymerase provided in trans; and (iii) apharmaceutically acceptable excipient.

Embodiment 286. A kit comprising a nucleic acid construct, comprising:(i) a nucleic acid sequencing encoding the fusion protein of any one ofembodiments 213-242; and (ii) a promoter that drives expression of thesequence of (i).

Embodiment 287. A method for installing a desired nucleotide change in adouble-stranded DNA sequence, the method comprising:

-   -   (i) contacting the double-stranded DNA sequence with a complex        comprising a fusion protein and a PEgRNA, wherein the fusion        protein comprises a napDNAbp and a polymerase and wherein the        PEgRNA comprises a DNA synthesis template comprising the desired        nucleotide change and a primer binding site;    -   (ii) nicking the double-stranded DNA sequence, thereby        generating a free single-strand DNA having a 3′ end;    -   (iii) hybridizing the 3′ end of the free single-strand DNA to        the primer binding site, thereby priming the polymerase;    -   (iv) polymerizing a strand of DNA from the 3′ end hybridized to        the primer binding site, thereby generating a single-strand DNA        flap comprising the desired nucleotide change and which is        complementary to the DNA synthesis template;    -   (v) replacing an endogenous DNA strand adjacent the cut site        with the single-strand DNA flap, thereby installing the desired        nucleotide change in the double-stranded DNA sequence.

Embodiment 288. The method of embodiment 287, wherein the step of (v)replacing comprises: (i) hybridizing the single-strand DNA flap to theendogenous DNA strand adjacent the cut site to create a sequencemismatch; (ii) excising the endogenous DNA strand; and (iii) repairingthe mismatch to form the desired product comprising the desirednucleotide change in both strands of DNA.

Embodiment 289. The method of embodiment 288, wherein the desirednucleotide change is a single nucleotide substitution, a deletion, or aninsertion.

Embodiment 290. The method of embodiment 289, wherein the singlenucleotide substitution is a transition or a transversion.

Embodiment 291. The method of embodiment 288, wherein the desirednucleotide change is (1) a G to T substitution, (2) a G to Asubstitution, (3) a G to C substitution, (4) a T to G substitution, (5)a T to A substitution, (6) a T to C substitution, (7) a C to Gsubstitution, (8) a C to T substitution, (9) a C to A substitution, (10)an A to T substitution, (11) an A to G substitution, or (12) an A to Csubstitution.

Embodiment 292. The method of embodiment 288, wherein the desirednucleoid change converts (1) a G:C basepair to a T:A basepair, (2) a G:Cbasepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) aT:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair,(6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:Cbasepair, (8) a C:G basepair to a T:A basepair, a C:G basepair to an A:Tbasepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepairto a G:C basepair, or (12) an A:T basepair to a C:G basepair.

Embodiment 293. The method of embodiment 288, wherein the desirednucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25nucleotides.

Embodiment 294. The method of embodiment 288, wherein the desirednucleotide change corrects a disease-associated gene.

Embodiment 295. The method of embodiment 294, wherein thedisease-associated gene is associated with a monogenetic disorderselected from the group consisting of: Adenosine Deaminase (ADA)Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; DuchenneMuscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease;Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1;Pachyonychia Congenita; Phenylkeotnuria; Severe CombinedImmunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; atrinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.

Embodiment 296. The method of embodiment 294, wherein thedisease-associated gene is associated with a polygenic disorder selectedfrom the group consisting of: heart disease; high blood pressure;Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 297. The method of embodiment 287, wherein the napDNAbp is anuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease activeCas9.

Embodiment 298. The method of embodiment 287, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18, or an amino acidsequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with the amino acid sequence of SEQ ID NO: 18.

Embodiment 299. The method of embodiment 287, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 300. The method of embodiment 287, wherein the polymerase isa reverse transcriptase comprising any one of the amino acid sequencesof SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159,235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 301. The method of embodiment 287, wherein the polymerase isa reverse transcriptase comprising an amino acid sequence having atleast 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the aminoacid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132,139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741,and 766.

Embodiment 302. The method of embodiment 287, wherein the PEgRNAcomprises a nucleic acid extension arm at the 3′ or 5′ ends or at anintramolecular location in the guide RNA, wherein the extension armcomprises the DNA synthesis template sequence and the primer bindingsite.

Embodiment 303. The method of embodiment 302, wherein the extension armis at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, atleast 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides,at least 19 nucleotides, at least 20 nucleotides, at least 21nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least24 nucleotides, or at least 25 nucleotides in length.

Embodiment 304. The method of embodiment 287, wherein the PEgRNA has anucleotide sequence selected from the group consisting of SEQ ID NOs:101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346,348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761,776-777.

Embodiment 305. A method for introducing one or more changes in thenucleotide sequence of a DNA molecule at a target locus, comprising:

-   -   (i) contacting the DNA molecule with a nucleic acid programmable        DNA binding protein (napDNAbp) and a PEgRNA which targets the        napDNAbp to the target locus, wherein the PEgRNA comprises a        reverse transcriptase (RT) template sequence comprising at least        one desired nucleotide change and a primer binding site;    -   (ii) forming an exposed 3′ end in a DNA strand at the target        locus;    -   (iii) hybridizing the exposed 3′ end to the primer binding site        to prime reverse transcription;    -   (iv) synthesizing a single strand DNA flap comprising the at        least one desired nucleotide change based on the RT template        sequence by reverse transcriptase;    -   (v) and incorporating the at least one desired nucleotide change        into the corresponding endogenous DNA, thereby introducing one        or more changes in the nucleotide sequence of the DNA molecule        at the target locus.

Embodiment 306. The method of embodiment 305, wherein the one or morechanges in the nucleotide sequence comprises a transition.

Embodiment 307. The method of embodiment 306, wherein the transition isselected from the group consisting of: (a) T to C; (b) A to G; (c) C toT; and (d) G to A.

Embodiment 308. The method of embodiment 305, wherein the one or morechanges in the nucleotide sequence comprises a transversion.

Embodiment 309. The method of embodiment 308, wherein the transversionis selected from the group consisting of: (a) T to A; (b) T to G; (c) Cto G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.

Embodiment 310. The method of embodiment 305, wherein the one or morechanges in the nucleotide sequence comprises changing (1) a G:C basepairto a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:Cbasepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) aT:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair,(7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:Abasepair, (9) a C:G basepair to an A:T basepair, (9) an A:T basepair toa T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:Tbasepair to a C:G basepair.

Embodiment 311. The method of embodiment 305, wherein the one or morechanges in the nucleotide sequence comprises an insertion or deletion of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25 nucleotides.

Embodiment 312. The method of embodiment 305, wherein the one or morechanges in the nucleotide sequence comprises a correction to adisease-associated gene.

Embodiment 313. The method of embodiment 312, wherein thedisease-associated gene is associated with a monogenetic disorderselected from the group consisting of: Adenosine Deaminase (ADA)Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; DuchenneMuscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease;Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1;Pachyonychia Congenita; Phenylkeotnuria; Severe CombinedImmunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; atrinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.

Embodiment 314. The method of embodiment 312, wherein thedisease-associated gene is associated with a polygenic disorder selectedfrom the group consisting of: heart disease; high blood pressure;Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 315. The method of embodiment 305, wherein the napDNAbp is anuclease active Cas9 or variant thereof.

Embodiment 316. The method of embodiment 305, wherein the napDNAbp is anuclease inactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variantthereof.

Embodiment 317. The method of embodiment 305, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18, or an amino acidsequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with SEQ ID NO: 18.

Embodiment 318. The method of embodiment 305, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 319. The method of embodiment 305, wherein the reversetranscriptase is introduced in trans.

Embodiment 320. The method of embodiment 305, wherein the napDNAbpcomprises a fusion to a reverse transcriptase.

Embodiment 321. The method of embodiment 305, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 322. The method of embodiment 305, wherein the reversetranscriptase comprises an amino acid sequence having at least 80%, 85%,90%, 95%, 98%, or 99% sequence identity with the amino acid sequence ofany one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149,154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 323. The method of embodiment 305, wherein the step offorming an exposed 3′ end in the DNA strand at the target locuscomprises nicking the DNA strand with a nuclease.

Embodiment 324. The method of embodiment 323, wherein the nuclease isprovided is provided in trans.

Embodiment 325. The method of embodiment 305, wherein the step offorming an exposed 3′ end in the DNA strand at the target locuscomprises contacting the DNA strand with a chemical agent.

Embodiment 326. The method of embodiment 305, wherein the step offorming an exposed 3′ end in the DNA strand at the target locuscomprises introducing a replication error.

Embodiment 327. The method of embodiment 305, wherein the step ofcontacting the DNA molecule with the napDNAbp and the guide RNA forms anR-loop.

Embodiment 328. The method of embodiment 327, wherein the DNA strand inwhich the exposed 3′ end is formed is in the R-loop.

Embodiment 329. The method of embodiment 315, wherein the PEgRNAcomprises an extension arm that comprises the reverse transcriptase (RT)template sequence and the primer binding site.

Embodiment 330. The method of embodiment 329, wherein the extension armis at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at anintramolecular position in the guide RNA.

Embodiment 331. The method of embodiment 305, wherein the PEgRNA furthercomprises at least one additional structure selected from the groupconsisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer,or an RNA-protein recruitment domain.

Embodiment 332. The method of embodiment 305, wherein the PEgRNA furthercomprises a homology arm.

Embodiment 333. The method of embodiment 305, wherein the RT templatesequence is homologous to the corresponding endogenous DNA.

Embodiment 334. A method for introducing one or more changes in thenucleotide sequence of a DNA molecule at a target locus by target-primedreverse transcription, the method comprising: (a) contacting the DNAmolecule at the target locus with a (i) fusion protein comprising anucleic acid programmable DNA binding protein (napDNAbp) and a reversetranscriptase and (ii) a guide RNA comprising an RT template comprisinga desired nucleotide change; (b) conducting target-primed reversetranscription of the RT template to generate a single strand DNAcomprising the desired nucleotide change; and (c) incorporating thedesired nucleotide change into the DNA molecule at the target locusthrough a DNA repair and/or replication process.

Embodiment 335. The method of embodiment 334, wherein the RT template islocated at the 3′ end of the guide RNA, the 5′ end of the guide RNA, orat an intramolecular location in the guide RNA.

Embodiment 336. The method of embodiment 334, wherein the desirednucleotide change comprises a transition, a transversion, an insertion,or a deletion, or any combination thereof.

Embodiment 337. The method of embodiment 334, wherein the desirednucleotide change comprises a transition selected from the groupconsisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.

Embodiment 338. The method of claim 334, wherein the desired nucleotidechange comprises a transversion selected from the group consisting of:(a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C;(g) G to C; and (h) G to T.

Embodiment 339. The method of embodiment 334, wherein the desirednucleotide change comprises changing (1) a G:C basepair to a T:Abasepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair toC:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepairto an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:Gbasepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) aC:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair,(11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:Gbasepair.

Embodiment 340. A polynucleotide encoding the PEgRNA of any one ofembodiments 243-259.

Embodiment 341. A vector comprising the polynucleotide of embodiment340.

Embodiment 342. A cell comprising the vector of embodiment 341.

Embodiment 343. The fusion protein of embodiment 213, wherein thepolymerase is an error-prone reverse transcriptase.

Embodiment 344. A method for mutagenizing a DNA molecule at a targetlocus by target-primed reverse transcription, the method comprising: (a)contacting the DNA molecule at the target locus with a (i) fusionprotein comprising a nucleic acid programmable DNA binding protein(napDNAbp) and an error-prone reverse transcriptase and (ii) a guide RNAcomprising an RT template comprising a desired nucleotide change; (b)conducting target-primed reverse transcription of the RT template togenerate a mutagenized single strand DNA; and (c) incorporating themutagenized single strand DNA into the DNA molecule at the target locusthrough a DNA repair and/or replication process.

Embodiment 345. The method of any prior embodiment, wherein the fusionprotein comprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 346. The method of any prior embodiment, wherein the napDNAbpis a Cas9 nickase (nCas9).

Embodiment 347. The method of embodiment 344, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 348. The method of embodiment 344, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 349. The method of embodiment 344, wherein the step of (b)conducting target-primed reverse transcription comprises generating a 3′end primer binding sequence at the target locus that is capable ofpriming reverse transcription by annealing to a primer binding site onthe guide RNA.

Embodiment 350. A method for replacing a trinucleotide repeat expansionmutation in a target DNA molecule with a healthy sequence comprising ahealthy number of repeat trinucleotides, the method comprising: (a)contacting the DNA molecule at the target locus with a (i) fusionprotein comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a polymerase and (ii) a PEgRNA comprising DNA synthesistemplate comprising the replacement sequence and a primer binding site;(b) conducting prime editing to generate a single strand DNA comprisingthe replacement sequence; and (c) incorporating the single strand DNAinto the DNA molecule at the target locus through a DNA repair and/orreplication process.

Embodiment 351. The method of embodiment 350, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 352. The method of embodiment 350, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 353. The method of embodiment 350, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 354. The method of embodiment 350, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 355. The method of embodiment 350, wherein the step of (b)conducting prime editing comprises generating a 3′ end primer bindingsequence at the target locus that is capable of priming polymerase byannealing to the primer binding site on the guide RNA.

Embodiment 356. The method of embodiment 350, wherein the trinucleotiderepeat expansion mutation is associated with Huntington's Disease,Fragile X syndrome, or Friedreich's ataxia.

Embodiment 357. The method of embodiment 350, wherein the trinucleotiderepeat expansion mutation comprises a repeating unit of CAG triplets.

Embodiment 358. The method of embodiment 350, wherein the trinucleotiderepeat expansion mutation comprises a repeating unit of GAA triplets.

Embodiment 359. A method of installing a functional moiety in a proteinof interest encoded by a target nucleotide sequence by prime editing,the method comprising: (a) contacting the target nucleotide sequencewith a (i) prime editor comprising a nucleic acid programmable DNAbinding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprisingDNA synthesis template encoding the functional moiety; (b) polymerizinga single strand DNA sequence encoding the functional moiety; and (c)incorporating the single strand DNA sequence in place of a correspondingendogenous strand at the target nucleotide sequence through a DNA repairand/or replication process, wherein the method produces a recombinanttarget nucleotide sequence that encodes a fusion protein comprising theprotein of interest and the functional moiety.

Embodiment 360. The method of embodiment 359, wherein functional moietyis peptide tag.

Embodiment 361. The method of embodiment 360, wherein the peptide tag isan affinity tag, solubilization tag, chromatography tag, epitope orimmunoepitope tag, or a fluorescence tag.

Embodiment 362. The method of embodiment 360, wherein the peptide tag isselected from the group consisting of: AviTag (SEQ ID NO: 245); C-tag(SEQ ID NO: 246); Calmodulin-tag (SEQ ID NO: 247); polyglutamate tag(SEQ ID NO: 248); E-tag (SEQ ID NO: 249); FLAG-tag (SEQ ID NO: 2);HA-tag (SEQ ID NO: 5); His-tag (SEQ ID NOs: 252-262); Myc-tag (SEQ IDNO: 6); NE-tag (SEQ ID NO: 264); Rho1D4-tag (SEQ ID NO: 265); S-tag (SEQID NO: 266); SBP-tag (SEQ ID NO: 267); Softag-1 (SEQ ID NO: 268);Softag-2 (SEQ ID NO: 269); Spot-tag (SEQ ID NO: 270); Strep-tag (SEQ IDNO: 271); TC tag (SEQ ID NO: 272); Ty tag (SEQ ID NO: 273); V5 tag (SEQID NO: 3); VSV-tag (SEQ ID NO: 275); and Xpress tag (SEQ ID NO: 276).

Embodiment 363. The method of embodiment 360, wherein the peptide tag isselected from the group consisting of: AU1 epitope (SEQ ID NO: 278); AU5epitope (SEQ ID NO: 279); Bacteriophage T7 epitope (T7-tag) (SEQ ID NO:280); Bluetongue virus tag (B-tag) (SEQ ID NO: 281); E2 epitope (SEQ IDNO: 282); Histidine affinity tag (HAT) (SEQ ID NO: 283); HSV epitope(SEQ ID NO: 284); Polyarginine (Arg-tag) (SEQ ID NO: 285); Polyaspartate(Asp-tag) (SEQ ID NO: 286); Polyphenylalanine (Phe-tag) (SEQ ID NO:287); Si-tag (SEQ ID NO: 288); S-tag (SEQ ID NO: 266); and VSV-G (SEQ IDNO: 275).

Embodiment 364. The method of embodiment 359, wherein the functionalmoiety is an immunoepitope.

Embodiment 365. The method of embodiment 364, wherein the immunoepitopeis selected from the group consisting of: tetanus toxoid (SEQ ID NO:396); diphtheria toxin mutant CRM197 (SEQ ID NO: 630); mumpsimmunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO:402); mumps immunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO:406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410);TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopestoward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward classI HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO:422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ IDNO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQID NO: 426).

Embodiment 366. The method of embodiment 359, wherein the functionalmoiety alters the localization of the protein of interest.

Embodiment 367. The method of embodiment 359, wherein the functionalmoiety is a degradation tag such that the degradation rate of theprotein of interest is altered.

Embodiment 368. The method of embodiment 367, wherein the degradationtag results in the elimination of the tagged protein.

Embodiment 369. The method of embodiment 359, wherein the functionalmoiety is a small molecule binding domain.

Embodiment 370. The method of embodiment 359, wherein the small moleculebinding domain is FKBP12 of SEQ ID NO: 488.

Embodiment 371. The method of embodiment 359, wherein the small moleculebinding domain is FKBP12-F36V of SEQ ID NO: 489.

Embodiment 372. The method of embodiment 359, wherein the small moleculebinding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.

Embodiment 373. The method of embodiment 359, wherein the small moleculebinding domain is installed in two or more proteins of interest.

Embodiment 374. The method of embodiment 373, wherein the two or moreproteins of interest may dimerize upon contacting with a small molecule.

Embodiment 375. The method of embodiment 369, wherein the small moleculeis a dimer of a small molecule selected from the group consisting ofthose compounds disclosed in Embodiment 163 of Group 1.

Embodiment 376. A method of installing an immunoepitope in a protein ofinterest encoded by a target nucleotide sequence by prime editing, themethod comprising: (a) contacting the target nucleotide sequence with a(i) prime editor comprising a nucleic acid programmable DNA bindingprotein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edittemplate encoding the functional moiety; (b) polymerizing a singlestrand DNA sequence encoding the immunoepitope; and (c) incorporatingthe single strand DNA sequence in place of a corresponding endogenousstrand at the target nucleotide sequence through a DNA repair and/orreplication process, wherein the method produces a recombinant targetnucleotide sequence that encodes a fusion protein comprising the proteinof interest and the immunoepitope.

Embodiment 377. The method of embodiment 376, wherein the immunoepitopeis selected from the group consisting of: tetanus toxoid (SEQ ID NO:396); diphtheria toxin mutant CRM197 (SEQ ID NO: 630); mumpsimmunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO:402); mumps immunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO:406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410);TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopestoward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward classI HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO:422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ IDNO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQID NO: 426).

Embodiment 378. The method of embodiment 376, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 379. The method of embodiment 376, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 380. The method of embodiment 376, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 381. The method of embodiment 376, wherein the PEgRNAcomprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 499-505, 735-761, 776-777.

Embodiment 382. A method of installing a small molecule dimerizationdomain in a protein of interest encoded by a target nucleotide sequenceby prime editing, the method comprising: (a) contacting the targetnucleotide sequence with a (i) prime editor comprising a nucleic acidprogrammable DNA binding protein (napDNAbp) and a polymerase and (ii) aPEgRNA comprising an edit template encoding the small moleculedimerization domain; (b) polymerizing a single strand DNA sequenceencoding the immunoepitope; and (c) incorporating the single strand DNAsequence in place of a corresponding endogenous strand at the targetnucleotide sequence through a DNA repair and/or replication process,wherein the method produces a recombinant target nucleotide sequencethat encodes a fusion protein comprising the protein of interest and thesmall molecule dimerization domain.

Embodiment 383. The method of embodiment 382, further comprisingconducting the method on a second protein of interest.

Embodiment 384. The method of embodiment 383, wherein the first proteinof interest and the second protein of interest dimerize in the presenceof a small molecule that binds to the dimerization domain on each ofsaid proteins.

Embodiment 385. The method of embodiment 382, wherein the small moleculebinding domain is FKBP12 of SEQ ID NO: 488.

Embodiment 386. The method of embodiment 382, wherein the small moleculebinding domain is FKBP12-F36V of SEQ ID NO: 489.

Embodiment 387. The method of embodiment 382, wherein the small moleculebinding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.

Embodiment 388. The method of embodiment 382, wherein the small moleculeis a dimer of a small molecule selected from the group consisting ofthose compounds disclosed in Embodiment 163 of Group 1.

Embodiment 389. The method of embodiment 382, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 390. The method of embodiment 382, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 391. The method of embodiment 382, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 392. The method of embodiment 382, wherein the PEgRNAcomprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 499-505, 735-761, 776-777.

Embodiment 393. A method of installing a peptide tag or epitope onto aprotein using prime editing, comprising: contacting a target nucleotidesequence encoding the protein with a prime editor construct configuredto insert therein a second nucleotide sequence encoding the peptide tagto result in a recombinant nucleotide sequence, such that the peptidetag and the protein are expressed from the recombinant nucleotidesequence as a fusion protein.

Embodiment 394. The method of embodiment 383, wherein the peptide tag isused for purification and/or detection of the protein.

Embodiment 395. The method of embodiment 383, wherein the peptide tag isa poly-histidine (e.g., HHHHHH) (SEQ ID NO: 252-262), FLAG (e.g.,DYKDDDDK) (SEQ ID NO: 2), V5 (e.g., GKPIPNPLLGLDST) (SEQ ID NO: 3),GCN4, HA (e.g., YPYDVPDYA) (SEQ ID NO: 5), Myc (e.g. EQKLISEED) (SEQ IDNO: 6), or GST.

Embodiment 396. The method of embodiment 383, wherein the peptide taghas an amino acid sequence selected from the group consisting of SEQ IDNO: 1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622.

Embodiment 397. The method of embodiment 383, wherein the peptide tag isfused to the protein by a linker.

Embodiment 398. The method of embodiment 383, wherein the fusion proteinhas the following structure: [protein]-[peptide tag] or [peptidetag]-[protein], wherein “]-[” represents an optional linker.

Embodiment 399. The method of embodiment 383, wherein the linker has anamino acid sequence of SEQ ID NO: 127, 165-176, 446,453, and 767-769.

Embodiment 400. The method of embodiment 383, wherein the prime editorconstruct comprises a PEgRNA comprising the nucleotide sequence of SEQID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 401. The method of embodiment 383, wherein the PEgRNAcomprises a spacer, a gRNA core, and an extension arm, wherein thespacer is complementary to the target nucleotide sequence and theextension arm comprises a reverse transcriptase template that encodesthe peptide tag.

Embodiment 402. The method of embodiment 383, wherein the PEgRNAcomprises a spacer, a gRNA core, and an extension arm, wherein thespacer is complementary to the target nucleotide sequence and theextension arm comprises a reverse transcriptase template that encodesthe peptide tag.

Embodiment 403. A method of preventing or halting the progression of aprion disease by installing on or more protective mutations into PRNPencoded by a target nucleotide sequence by prime editing, the methodcomprising: (a) contacting the target nucleotide sequence with a (i)prime editor comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edittemplate encoding the functional moiety; (b) polymerizing a singlestrand DNA sequence encoding the protective mutation; and (c)incorporating the single strand DNA sequence in place of a correspondingendogenous strand at the target nucleotide sequence through a DNA repairand/or replication process, wherein the method produces a recombinanttarget nucleotide sequence that encodes a PRNP comprising a protectivemutation and which is resistant to misfolding.

Embodiment 404. The method of embodiment 403, wherein the prion diseaseis a human prion disease.

Embodiment 405. The method of embodiment 403, wherein the prion diseaseis an animal prion disease.

Embodiment 406. The method of embodiment 404, wherein the prion diseaseis Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease(vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal FamilialInsomnia, or Kuru.

Embodiment 407. The method of embodiment 403, wherein the prion diseaseis Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), ChronicWasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy,Feline Spongiform Encephalopathy, and Ungulate SpongiformEncephalopathy.

Embodiment 408. The method of embodiment 403, wherein the wildtype PRNPamino acid sequence is SEQ ID NOs: 291-292.

Embodiment 409. The method of embodiment 403, wherein the method resultsin a modified PRNP amino acid sequence selected from the groupconsisting of SEQ ID NOs: 293-309, 311-323, wherein said modified PRNPprotein is resistant to misfolding.

Embodiment 410. The method of embodiment 403, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 411. The method of embodiment 403, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 412. The method of embodiment 403, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 413. The method of embodiment 403, wherein the PEgRNAcomprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 499-505, 735-761, 776-777.

Embodiment 414. A method of installing a ribonucleotide motif or tag inan RNA of interest encoded by a target nucleotide sequence by primeediting, the method comprising: (a) contacting the target nucleotidesequence with a (i) prime editor comprising a nucleic acid programmableDNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNAcomprising an edit template encoding the ribonucleotide motif or tag;(b) polymerizing a single strand DNA sequence encoding theribonucleotide motif or tag; and (c) incorporating the single strand DNAsequence in place of a corresponding endogenous strand at the targetnucleotide sequence through a DNA repair and/or replication process,wherein the method produces a recombinant target nucleotide sequencethat encodes a modified RNA of interest comprising the ribonucleotidemotif or tag.

Embodiment 415. The method of embodiment 414, wherein ribonucleotidemotif or tag is a detection moiety.

Embodiment 416. The method of embodiment 414, wherein the ribonucleotidemotif or tag affects the expression level of the RNA of interest.

Embodiment 417. The method of embodiment 414, wherein the ribonucleotidemotif or tag affects the transport or subcellular location of the RNA ofinterest.

Embodiment 418. The method of embodiment 414, wherein the ribonucleotidemotif or tag is selected from the group consisting of SV40 type 1, SV40type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PANENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer,spinach aptamer, mango aptamer, HDV ribozyme, and m6A.

Embodiment 419. The method of embodiment 414, wherein the PEgRNAcomprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 499-505, 735-761, 776-777.

Embodiment 420. The method of embodiment 414, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 421. The method of embodiment 414, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 422. The method of embodiment 414, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 423. A method of installing or deleting a functional moietyin a protein of interest encoded by a target nucleotide sequence byprime editing, the method comprising: (a) contacting the targetnucleotide sequence with a (i) prime editor comprising a nucleic acidprogrammable DNA binding protein (napDNAbp) and a polymerase and (ii) aPEgRNA comprising an edit template encoding the functional moiety ordeletion of same; (b) polymerizing a single strand DNA sequence encodingthe functional moiety or deletion of same; and (c) incorporating thesingle strand DNA sequence in place of a corresponding endogenous strandat the target nucleotide sequence through a DNA repair and/orreplication process, wherein the method produces a recombinant targetnucleotide sequence that encodes a modified protein comprising theprotein of interest and the functional moiety or the removal of same,wherein the functional moiety alters a modification state orlocalization state of the protein.

Embodiment 424. The method of embodiment 423, wherein functional moietyalters the phosphorylation, ubiquitylation, glycosylation, lipidation,hydroxylation, methylation, acetylation, crotonylation, SUMOylationstate of the protein of interest.

Embodiment 425. A fusion protein comprising a nucleic acid programmableDNA binding protein (napDNAbp) domain and a domain comprising anRNA-dependent DNA polymerase activity.

Embodiment 426. The fusion protein of embodiment 425, wherein the fusionprotein is capable of carrying out prime editing in the presence of anprime editing guide RNA (PEgRNA) to install a desired nucleotide changein a target sequence.

Embodiment 427. The fusion protein of embodiment 425, wherein thenapDNAbp domain has a nickase activity.

Embodiment 428. The fusion protein of embodiment 425, wherein thenapDNAbp domain is a Cas9 protein or variant thereof.

Embodiment 429. The fusion protein of embodiment 425, wherein thenapDNAbp domain is a nuclease active Cas9, a nuclease inactive Cas9(dCas9), or a Cas9 nickase (nCas9).

Embodiment 430. The fusion protein of embodiment 425, wherein thenapDNAbp domain is Cas9 nickase (nCas9).

Embodiment 431. The fusion protein of embodiment 425, wherein thenapDNAbp domain is selected from the group consisting of: Cas9, Cas12e,Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionallyhas a nickase activity.

Embodiment 432. The fusion protein of embodiment 425, wherein the domaincomprising an RNA-dependent DNA polymerase activity is a reversetranscriptase comprising any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 433. The fusion protein of embodiment 425, wherein the domaincomprising an RNA-dependent DNA polymerase activity is a reversetranscriptase comprising an amino acid sequence having at least 80%,85%, 90%, 95%, 98%, or 99% sequence identity with the amino acidsequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139,143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and766, and optionally wherein the domain comprising an RNA-dependent DNApolymerase is error-prone.

Embodiment 434. The fusion protein of embodiment 425, wherein the domaincomprising an RNA-dependent DNA polymerase activity is anaturally-occurring reverse transcriptase from a retrovirus or aretrotransposon.

Embodiment 435. The fusion protein of embodiment 425, wherein the fusionprotein when complexed with a PEgRNA is capable of binding to a targetDNA sequence.

Embodiment 436. The fusion protein of embodiment 435, wherein the targetDNA sequence comprises a target strand and a complementary non-targetstrand.

Embodiment 437. The fusion protein of embodiment 435, wherein thebinding of the fusion protein complexed to the PEgRNA forms an R-loop.

Embodiment 438. The fusion protein of embodiment 437, wherein the R-loopcomprises (i) an RNA-DNA hybrid comprising the PEgRNA and the targetstrand, and (ii) the complementary non-target strand.

Embodiment 439. The fusion protein of embodiment 437, wherein the targetor the complementary non-target strand is nicked to form a primingsequence having a free 3′ end.

Embodiment 440. The fusion protein of embodiment 439, wherein the nicksite is upstream of the PAM sequence on the target strand.

Embodiment 441. The fusion protein of embodiment 439, wherein the nicksite is upstream of the PAM sequence on the non-target strand.

Embodiment 442. The fusion protein of embodiment 439, wherein the nicksite-1, -2, -3, -4, -5, -6, -7, -8, or -9 relative to the 5′ end of thePAM sequence.

Embodiment 443. The fusion protein of embodiment 426, wherein the PEgRNAcomprises a guide RNA and at least one nucleic acid extension arm.

Embodiment 444. The fusion protein of embodiment 443, wherein theextension arm is at the 5′ or the 3′ end of the guide RNA, or at anintramolecular location in the guide RNA.

Embodiment 445. The fusion protein of embodiment 443, wherein theextension arm comprises (i) a DNA synthesis template sequence comprisinga desired nucleotide change, and (ii) a primer binding site.

Embodiment 446. The fusion protein of embodiment 445, wherein the DNAsynthesis template sequence encodes a single-strand DNA flap that iscomplementary to an endogenous DNA sequence adjacent to the nick site,wherein the single-strand DNA flap comprises the desired nucleotidechange.

Embodiment 447. The fusion protein of embodiment 443, wherein theextension arm is at least 5 nucleotides, at least 6 nucleotides, atleast 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, atleast 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides,at least 13 nucleotides, at least 14 nucleotides, at least 15nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, atleast 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides,at least 24 nucleotides, at least 25 nucleotides, at least 26nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, atleast 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides,at least 35 nucleotides, at least 36 nucleotides, at least 37nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least40 nucleotides, at least 41 nucleotides, at least 42 nucleotides, atleast 43 nucleotides, at least 44 nucleotides, at least 45 nucleotides,at least 46 nucleotides, at least 47 nucleotides, at least 48nucleotides, at least 49 nucleotides, or at least 50 nucleotides.

Embodiment 448. The fusion protein of embodiment 443, wherein thesingle-strand DNA flap hybridizes to the endogenous DNA sequenceadjacent to the nick site, thereby installing the desired nucleotidechange in the target strand.

Embodiment 449. The fusion protein of embodiment 443, wherein thesingle-stranded DNA flap displaces the endogenous DNA sequence adjacentto the nick site and which has a free 5′ end.

Embodiment 450. The fusion protein of embodiment 446, wherein theendogenous DNA sequence having the 5′ end is excised by the cell.

Embodiment 451. The fusion protein of embodiment 446, wherein theendogenous DNA sequence having the 5′ end is excised by a flapendonuclease.

Embodiment 452. The fusion protein of embodiment 448, wherein cellularrepair of the single-strand DNA flap incorporates the desired nucleotidechange in the non-target strand, thereby forming a desired product.

Embodiment 453. The fusion protein of embodiment 449, wherein thedesired nucleotide change is installed in an editing window that isbetween about −4 to +10 of the PAM sequence, or between about −10 to +20of the PAM sequence, or between about −20 to +40 of the PAM sequence, orbetween about −30 to +100 of the PAM sequence.

Embodiment 454. The fusion protein of embodiment 449, wherein thedesired nucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100 nucleotides downstream of the nick site.

Embodiment 455. The fusion protein of embodiment 425, wherein thenapDNAbp comprises an amino acid sequence of SEQ ID NO:18, or an aminoacid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with the amino acid sequence to SEQ ID NO: 18.

Embodiment 456. The fusion protein of embodiment 425, wherein thenapDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%,95%, 98%, or 99% sequence identity with the amino acid sequence of anyone of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460,467, and 482-487.

Embodiment 457. The fusion protein of any one of the previousembodiments, wherein the fusion protein comprises the structureNH₂-[napDNAbp]-[domain comprising an RNA-dependent DNA polymeraseactivity]-COOH; or NH₂-[domain comprising an RNA-dependent DNApolymerase activity]-[napDNAbp]-COOH, wherein each instance of “]-[”indicates the presence of an optional linker sequence.

Embodiment 458. The fusion protein of embodiment 457, wherein the linkersequence comprises an amino acid sequence of SEQ ID NOs: 127, 165-176,446, 453, and 767-769.

Embodiment 459. The fusion protein of embodiment 425, wherein thedesired nucleotide change is a single nucleotide change, an insertion ofone or more nucleotides, or a deletion of one or more nucleotides.

Embodiment 460. The fusion protein of embodiment 459, wherein the insertor deletion is at least 1, at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33, at least 34, at least 35, at least 36, at least 37, at least 38, atleast 39, at least 40, at least 41, at least 42, at least 43, at least44, at least 45, at least 46, at least 47, at least 48, at least 49, orat least 50.

Embodiment 461. A complex comprising a fusion protein of any ofembodiments 425-460 and a PEgRNA, wherein the PEgRNA directs the fusionprotein to a target DNA sequence for prime editing.

Embodiment 462. The complex of embodiment 461, wherein the PEgRNAcomprises a guide RNA and a nucleic acid extension arm at the 3′ or 5′end of the guide RNA or at an intramolecular position in the guide RNA.

Embodiment 463. The complex of embodiment 462, wherein the PEgRNA iscapable of binding to a napDNAbp and directing the napDNAbp to thetarget DNA sequence.

Embodiment 464. The complex of embodiment 463, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand, wherein the guide RNA hybridizes to the target strand to form anRNA-DNA hybrid and an R-loop.

Embodiment 465. The complex of embodiment 464, wherein the at least onenucleic acid extension arm comprises (i) a DNA synthesis template, and(ii) a primer binding site.

Embodiment 466. The complex of embodiment 464, wherein the PEgRNAcomprises the nucleotide sequence of SEQ ID NOs: 101-104, 181-183,223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or anucleotide sequence having at least 85%, or at least 90%, or at least95%, or at least 98%, or at least 99% sequence identity with any one ofSEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 467. The complex of embodiment 465, wherein the DNA synthesistemplate comprises a nucleotide sequence that is at least 80%, or 85%,or 90%, or 95%, or 99% identical to the endogenous DNA target.

Embodiment 468. The complex of embodiment 465, wherein the primerbinding site hybridizes with a free 3′ end of the cut DNA.

Embodiment 469. The complex of embodiment 461, wherein the napDNAbp is aCas9 nickase.

Embodiment 470. The complex of embodiment 461, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18, or an amino acidsequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with SEQ ID NO: 18.

Embodiment 471. The complex of embodiment 461, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 472. The complex of embodiment 461, wherein the PEgRNAcomprises a guide RNA and a nucleic acid extension arm at the 3′ or 5′end of the guide RNA, or at an intramolecular position in the guide RNA.

Embodiment 473. The complex of embodiment 465, wherein the DNA synthesistemplate comprises a nucleotide sequence that is at least 80%, or 85%,or 90%, or 95%, or 99% identical to the endogenous DNA target.

Embodiment 474. The complex of embodiment 465, wherein the primerbinding site hybridizes with a free 3′ end of the cut DNA.

Embodiment 475. The complex of embodiment 462, wherein the PEgRNAfurther comprises at least one additional structure selected from thegroup consisting of a linker, a stem loop, a hairpin, a toeloop, anaptamer, or an RNA-protein recruitment domain.

Embodiment 476. A polynucleotide encoding the fusion protein of any ofembodiments 425-461.

Embodiment 477. A polynucleotide encoding the PEgRNA of any of the aboveembodiments.

Embodiment 478. A vector comprising the polynucleotide of embodiment476, wherein expression of the fusion protein is under the control of apromoter.

Embodiment 479. A vector comprising the polynucleotide of embodiment477, wherein expression of the PEgRNA is under the control of apromoter.

Embodiment 480. The vector of embodiment 479, wherein the promoter is aU6 promoter.

Embodiment 481. The vector of embodiment 479, wherein the promoter is aCMV promoter.

Embodiment 482. The vector of embodiment 480, wherein the PEgRNA isengineered to remove one or more repeating clusters of Ts in theextension arm to improve transcription efficiency by the U6 promoter.

Embodiment 483. The vector of embodiment 482, wherein the one or morerepeating clusters of Ts that is removed comprises at least 3 Ts, atleast 4 Ts, at least 5 Ts, at least 6 Ts, at least 7 Ts, at least 8 Ts,at least 9 Ts, at least 10 Ts, at least 11 Ts, at least 12 Ts, at least13 Ts, at least 14 Ts, at least 15 Ts, at least 16 Ts, at least 17 Ts,at least 18 Ts, at least 19 Ts, or at least 20 Ts.

Embodiment 484. A cell comprising the fusion protein of any ofembodiments 425-460 and an PEgRNA bound to the napDNAbp of the fusionprotein.

Embodiment 485. A cell comprising a complex of any one of embodiments461-475.

Embodiment 486. A pharmaceutical composition comprising: (i) a fusionprotein of any of embodiments 425-460, the complex of embodiments461-475, the polynucleotide of embodiments 476-477, or the vector ofembodiments 478-483; and (ii) a pharmaceutically acceptable excipient.

Embodiment 487. A pharmaceutical composition comprising: (i) the complexof embodiments 461-475 (ii) a polymerase provided in trans; and (iii) apharmaceutically acceptable excipient.

Embodiment 488. A kit for prime editing comprising: (i) a nucleic acidmolecule encoding the fusion protein of any one of embodiments 425-460;and (ii) a nucleic acid molecule encoding a PEgRNA that is capable ofdirecting the fusion protein to a target DNA site, wherein the nucleicacid molecule of (i) and (ii) may be contained within a single DNAconstruct or separate DNA constructs.

Embodiment 489. The kit of embodiment 488, wherein the nucleic acidmolecule of (i) further comprising a promoter that drives expression ofthe fusion protein.

Embodiment 490. The kit of embodiment 488, wherein the nucleic acidmolecule of (ii) further comprises a promoter that drives expression ofthe PEgRNA.

Embodiment 491. The kit of embodiment 490, wherein the promoter is a U6promoter.

Embodiment 492. The kit of embodiment 490, wherein the promoter is a CMVpromoter.

Embodiment 493. The fusion protein of embodiment 457, wherein the linkersequence comprises an amino acid sequence of SEQ ID NOs: 174 (1×SGGS),446 (2x SGGS), 3889 (3×SGGS), 171 (1×XTEN), 3968 (1×EAAAK), 3969(2×EAAAK), and 3970 (3×EAAAK).

GROUP B. PE GUIDES and METHODS OF DESIGN

Embodiment 1. A PEgRNA comprising a guide RNA and at least one nucleicacid extension arm comprising a DNA synthesis template.

Embodiment 2. The PEgRNA of embodiment 1, wherein the nucleic acidextension arm is position at the 3′ or 5′ end of the guide RNA, or at anintramolecular position in the guide RNA, and wherein the nucleic acidextension arm is DNA or RNA.

Embodiment 3. The PEgRNA of embodiment 1, wherein the PEgRNA is capableof binding to a napDNAbp and directing the napDNAbp to a target DNAsequence.

Embodiment 4. The PEgRNA of embodiment 3, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand.

Embodiment 5. The PEgRNA of embodiment 3, wherein the guide RNAhybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.

Embodiment 6. The PEgRNA of embodiment 1, wherein the at least onenucleic acid extension arm further comprises a primer binding site.

Embodiment 7. The PEgRNA of embodiment 1, wherein the nucleic acidextension arm is at least 5 nucleotides, at least 6 nucleotides, atleast 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, atleast 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides,at least 13 nucleotides, at least 14 nucleotides, at least 15nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, atleast 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides,at least 24 nucleotides, at least 25 nucleotides, at least 26nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, atleast 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides,at least 35 nucleotides, at least 36 nucleotides, at least 37nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least40 nucleotides, at least 41 nucleotides, at least 42 nucleotides, atleast 43 nucleotides, at least 44 nucleotides, at least 45 nucleotides,at least 46 nucleotides, at least 47 nucleotides, at least 48nucleotides, at least 49 nucleotides, or at least 50 nucleotides.

Embodiment 8. The PEgRNA of embodiment 1, wherein the DNA synthesistemplate is at least 3 nucleotides, at least 4 nucleotides, at least 5nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, or at least 15 nucleotides in length.

Embodiment 9. The PEgRNA of embodiment 6, wherein the primer bindingsite is at least 3 nucleotides, at least 4 nucleotides, at least 5nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, or at least 15 nucleotides in length.

Embodiment 10. The PEgRNA of embodiment 1, further comprising at leastone additional structure selected from the group consisting of a tRNA,linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-proteinrecruitment domain.

Embodiment 11. The PEgRNA of embodiment 1, wherein the DNA synthesistemplate encodes a single-strand DNA flap that is complementary to anendogenous DNA sequence adjacent to a nick site, wherein thesingle-strand DNA flap comprises a desired nucleotide change.

Embodiment 12. The PEgRNA of embodiment 11, wherein the single-strandedDNA flap displaces an endogenous single-strand DNA having a 5′ end inthe target DNA sequence that has been nicked, and wherein the endogenoussingle-strand DNA is immediately adjacent downstream of the nick site.

Embodiment 13. The PEgRNA of embodiment 11, wherein the endogenoussingle-stranded DNA having the free 5′ end is excised by the cell.

Embodiment 14. The PEgRNA of embodiment 13, wherein cellular repair ofthe single-strand DNA flap results in installation of the desirednucleotide change, thereby forming a desired product.

Embodiment 15. The PEgRNA of embodiment 1, comprising the nucleotidesequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 499-505, 735-761, 776-777, or a nucleotide sequence having atleast 85%, or at least 90%, or at least 95%, or at least 98%, or atleast 99% sequence identity with any one of SEQ ID NOs: 101-104,181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.

Embodiment 16. The PEgRNA of embodiment 1, wherein the DNA synthesistemplate comprises a nucleotide sequence that is at least 80%, or 85%,or 90%, or 95%, or 99% identical to the endogenous DNA target.

Embodiment 17. The PEgRNA of embodiment 6, wherein the primer bindingsite hybridizes with a free 3′ end of the cut DNA.

Embodiment 18. The PEgRNA of embodiment 10, wherein the at least oneadditional structure is located at the 3′ or 5′ end of the PEgRNA.

Embodiment 19. The PEgRNA of embodiment 10, wherein the linker comprisesa nucleotide sequence selected from the group consisting of SEQ ID NOs:127, 165-176, 446, 453, and 767-769.

Embodiment 20. The PEgRNA of embodiment 10, wherein the stem loopcomprises a nucleotide sequence selected from the stem loops describedherein.

Embodiment 21. The PEgRNA of embodiment 10, wherein the hairpincomprises a nucleotide sequence selected from the hairpins describedherein.

Embodiment 22. The PEgRNA of embodiment 10, wherein the toeloopcomprises a nucleotide sequence selected from the toeloops describedherein.

Embodiment 23. The PEgRNA of embodiment 10, wherein the aptamercomprises a nucleotide sequence selected from the aptamers describedherein.

Embodiment 24. The PEgRNA of embodiment 10, wherein the RNA-proteinrecruitment domain comprises a nucleotide sequence selected from theRNA-protein recruitment domain described herein.

Embodiment 25. A method for designing a PEgRNA for use in prime editingto install a desired nucleotide change in a target nucleotide sequence,wherein said PEgRNA comprises a spacer, gRNA core, and an extension arm,and wherein said extension arm comprises a primer binding site and a DNAsynthesis template, said method comprising:

-   -   (i) selecting a desired target edit site in a target nucleotide        sequence;    -   (ii) obtaining a context nucleotide sequence upstream and        downstream from the target edit site;    -   (iii) locating putative protospacer adjacent motif (PAM) sites        in the context nucleotide sequence which are proximal to the        desired target edit site;    -   (iv) identifying the corresponding nick sites for each putative        PAM site;    -   (v) designing the spacer;    -   (vi) designing the gRNA core;    -   (vii) designing the extension arm; and    -   (viii) constructing the full PEgRNA by concatenating the spacer,        gRNA core, and the extension arm.

Embodiment 26. The method of embodiment 25, wherein the step (i) ofselecting the desired target edit site comprises selecting adisease-causing mutation.

Embodiment 27. The method of embodiment 26, wherein the disease-causingmutation is associated with a disease selected from the group consistingof: cancer, autoimmune disorders, neurological disorders, skindisorders, respiratory diseases, and cardiac diseases.

Embodiment 28. The method of embodiment 25, wherein the step (ii) ofobtaining a context nucleotide sequence upstream and downstream from thetarget edit site comprises obtaining about 50-55 base pairs (bp), about55-60 bp, about 60-65 bp, about 65-70 bp, about 70-75 bp, about 75-80bp, about 80-85 bp, about 85-90 bp, about 90-95 bp, about 95-100 bp,about 100-105 bp, about 105-110 bp, about 110-125 bp, about 125-130 bp,about 130-135 bp, about 135-140 bp, about 140-145 bp, about 145-150 bp,about 150-155 bp, about 155-160 bp, about 160-165 bp, about 165-170 bp,about 170-175 bp, about 175-180 bp, about 180-185 bp, about 185-190 bp,about 190-195 bp, about 195-200 bp, about 200-205 bp, about 205-210 bp,about 210-215 bp, about 215-220 bp, about 220-225 bp, about 225-230 bp,about 230-235 bp, about 235-240 bp, about 240-245 bp, or about 245-250bp of a region that comprises the desired target edit site.

Embodiment 29. The method of embodiment 28, wherein the desired targetedit site is positioned approximately equidistant from each end of thecontext nucleotide sequence.

Embodiment 30. The method of embodiment 25, wherein in step (iii), theputative PAM sites are proximal to the desired target edit site.

Embodiment 31. The method of embodiment 25, wherein in step (iii), theputative PAM sites comprise those with associated nick sites located ata position less than 30 nucleotides from the target edit site, or lessthan 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides from the target editsite.

Embodiment 32. The method of embodiment 25, wherein in step (iii), theputative PAM sites comprise those with associated nick sites located ata position more than 30 nucleotides from the target edit site, or morethan 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100nucleotides from the target edit site.

Embodiment 33. The method of embodiment 25, wherein in step (iii), theputative PAM sites are associated with one or corresponding napDNAbpwhich bind to said PAM sites.

Embodiment 34. The method of embodiment 33, the putative PAM sites andtheir corresponding napDNAbps are selected from any of the followinggroupings: (a) SpCas9 of SEQ ID NO: 18-25 and 87-88 and NGG; (b) Sp VQRnCas9; and (c) NGAN.

Embodiment 35. The method of embodiment 25, wherein the step (v) ofdesigning the spacer comprises determining the complement nucleotidesequence of the protospacer sequence associated with each putative PAM.

Embodiment 36. The method of embodiment 25, wherein the step (vi) ofdesigning gRNA core comprises in the context of each putative PAM,selecting a gRNA core sequence that is capable of binding to a napDNAbpwhich is associated with each of said putative PAMs.

Embodiment 37. The method of embodiment 25, wherein the step (vii) ofdesigning the extension arm comprises designing (a) a DNA synthesistemplate comprising the edit of interest, and (b) a primer binding site.

Embodiment 38. The method of embodiment 37, wherein designing the primerbinding site comprises (a) identifying a DNA primer on thePAM-containing strand of the target nucleotide sequence, wherein the 3′end of the DNA primer is the first nucleotide upstream of the nick siteassociated with the PAM site, and (b) designing the complement of theDNA primer, wherein said complement forms the primer binding site.

Embodiment 39. The method of embodiment 38, wherein the primer bindingsite is 8 to 15 nucleotides in length.

Embodiment 40. The method of embodiment 38, wherein the primer bindingsite is 12-13 nucleotides if the DNA primer contains about 40-60% GCcontent.

Embodiment 41. The method of embodiment 38, wherein the primer bindingsite is 14-15 nucleotides if the DNA primer contains less than about 40%GC content.

Embodiment 42. The method of embodiment 38, wherein the primer bindingsite is 8-11 nucleotides if the DNA primer contains greater than about60% GC content.

Embodiment 43. A method of prime editing comprising contacting a targetDNA sequence with a PEgRNA of any of embodiments 1-24 and a prime editorfusion protein comprising a napDNAbp and a domain having anRNA-dependent DNA polymerase activity.

Embodiment 44. The method of embodiment 43, wherein the napDNAbp has anickase activity.

Embodiment 45. The method of embodiment 43, wherein the napDNAbp is aCas9 protein or variant thereof.

Embodiment 46. The method of embodiment 43, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

Embodiment 47. The method of embodiment 43, wherein the napDNAbp is Cas9nickase (nCas9).

Embodiment 48. The method of embodiment 43, wherein the napDNAbp isselected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a,Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickaseactivity.

Embodiment 49. The method of embodiment 43, wherein the domaincomprising an RNA-dependent DNA polymerase activity is a reversetranscriptase comprising any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 50. The method of embodiment 43, wherein the domaincomprising an RNA-dependent DNA polymerase activity is a reversetranscriptase comprising an amino acid sequence having at least 80%,85%, 90%, 95%, 98%, or 99% sequence identity with the amino acidsequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139,143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and766.

Embodiment 51. The method of embodiment 43, wherein the domaincomprising an RNA-dependent DNA polymerase activity is anaturally-occurring reverse transcriptase from a retrovirus or aretrotransposon.

Group C. PE Complexes

Embodiment 1. A complex for prime editing comprising:

-   -   (i) fusion protein comprising a nucleic acid programmable DNA        binding protein (napDNAbp) and a domain comprising an        RNA-dependent DNA polymerase activity; and    -   (ii) a prime editing guide RNA (PEgRNA).

Embodiment 2. The complex of embodiment 1, wherein the fusion protein iscapable of carrying out prime editing in the presence of the primeediting guide RNA (PEgRNA) to install a desired nucleotide change in atarget sequence.

Embodiment 3. The complex of embodiment 11, wherein the napDNAbp has anickase activity.

Embodiment 4. The complex of embodiment 1, wherein the napDNAbp is aCas9 protein or variant thereof.

Embodiment 5. The complex of embodiment 1, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

Embodiment 6. The complex of embodiment 1, wherein the napDNAbp is Cas9nickase (nCas9).

Embodiment 7. The complex of embodiment 1, wherein the napDNAbp isselected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a,Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickaseactivity.

Embodiment 8. The complex of embodiment 1, wherein the domain comprisingan RNA-dependent DNA polymerase activity is a reverse transcriptasecomprising any one of the amino acid sequences of SEQ ID NOs: 89-100,105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662,700, 701-716, 739-741, and 766.

Embodiment 9. The complex of embodiment 1, wherein the domain comprisingan RNA-dependent DNA polymerase activity is a reverse transcriptasecomprising an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235,454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 10. The complex of embodiment 1, wherein the domaincomprising an RNA-dependent DNA polymerase activity is anaturally-occurring reverse transcriptase from a retrovirus or aretrotransposon.

Embodiment 11. The complex of embodiment 1, wherein the fusion proteinwhen complexed with a PEgRNA is capable of binding to a target DNAsequence.

Embodiment 12. The complex of embodiment 1, wherein the PEgRNA comprisesa guide RNA and at least one nucleic acid extension arm comprising a DNAsynthesis template.

Embodiment 13. The complex of embodiment 12, wherein the nucleic acidextension arm is position at the 3′ or 5′ end of the guide RNA, or at anintramolecular position in the guide RNA, and wherein the nucleic acidextension arm is DNA or RNA.

Embodiment 14. The complex of embodiment 12, wherein the PEgRNA iscapable of binding to a napDNAbp and directing the napDNAbp to a targetDNA sequence.

Embodiment 15. The complex of embodiment 14, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand.

Embodiment 16. The complex of embodiment 12, wherein the guide RNAhybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.

Embodiment 17. The complex of embodiment 12, wherein the at least onenucleic acid extension arm further comprises a primer binding site.

Embodiment 18. The complex of embodiment 12, wherein the nucleic acidextension arm is at least 5 nucleotides, at least 6 nucleotides, atleast 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, atleast 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides,at least 13 nucleotides, at least 14 nucleotides, at least 15nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, atleast 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides,at least 24 nucleotides, at least 25 nucleotides, at least 26nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, atleast 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides,at least 35 nucleotides, at least 36 nucleotides, at least 37nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least40 nucleotides, at least 41 nucleotides, at least 42 nucleotides, atleast 43 nucleotides, at least 44 nucleotides, at least 45 nucleotides,at least 46 nucleotides, at least 47 nucleotides, at least 48nucleotides, at least 49 nucleotides, or at least 50 nucleotides.

Embodiment 19. The complex of embodiment 12, wherein the DNA synthesistemplate is at least 3 nucleotides, at least 4 nucleotides, at least 5nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, or at least 15 nucleotides in length.

Embodiment 20. The complex of embodiment 17, wherein the primer bindingsite is at least 3 nucleotides, at least 4 nucleotides, at least 5nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, atleast 14 nucleotides, or at least 15 nucleotides in length.

Embodiment 21. The complex of embodiment 12, wherein the PEgRNA furthercomprises at least one additional structure selected from the groupconsisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer,or an RNA-protein recruitment domain.

Embodiment 22. The complex of embodiment 12, wherein the DNA synthesistemplate encodes a single-strand DNA flap that is complementary to anendogenous DNA sequence adjacent to a nick site, wherein thesingle-strand DNA flap comprises a desired nucleotide change.

Embodiment 23. The complex of embodiment 22, wherein the single-strandedDNA flap displaces an endogenous single-strand DNA having a 5′ end inthe target DNA sequence that has been nicked, and wherein the endogenoussingle-strand DNA is immediately adjacent downstream of the nick site.

Embodiment 24. The complex of embodiment 23, wherein the endogenoussingle-stranded DNA having the free 5′ end is excised by the cell.

Embodiment 25. The complex of embodiment 23, wherein cellular repair ofthe single-strand DNA flap results in installation of the desirednucleotide change, thereby forming a desired product.

Embodiment 26. The complex of embodiment 12, wherein the PEgRNAcomprises the nucleotide sequence of SEQ ID NOs: 101-104, 181-183,223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or anucleotide sequence having at least 85%, or at least 90%, or at least95%, or at least 98%, or at least 99% sequence identity with any one ofSEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 27. The complex of embodiment 12, wherein the DNA synthesistemplate comprises a nucleotide sequence that is at least 80%, or 85%,or 90%, or 95%, or 99% identical to the endogenous DNA target.

Embodiment 28. The complex of embodiment 17, wherein the primer bindingsite hybridizes with a free 3′ end of the cut DNA.

Embodiment 29. The complex of embodiment 21, wherein the at least oneadditional structure is located at the 3′ or 5′ end of the PEgRNA.

Embodiment 30. The complex of embodiment 29, wherein the linkercomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Embodiment 31. The complex of embodiment 29, wherein the stem loopcomprises a nucleotide sequence selected from the stem loops describedherein.

Embodiment 32. The complex of embodiment 29, wherein the hairpincomprises a nucleotide sequence selected from the hairpins describedherein.

Embodiment 33. The complex of embodiment 29, wherein the toeloopcomprises a nucleotide sequence selected from the toeloops describedherein.

Embodiment 34. The complex of embodiment 29, wherein the aptamercomprises a nucleotide sequence selected from the aptamers describedherein.

Embodiment 35. The complex of embodiment 29, wherein the RNA-proteinrecruitment domain comprises a nucleotide sequence selected from theRNA-protein recruitment domains described herein.

Embodiment 36. The complex of embodiment 1, wherein the target DNAsequence comprises a target strand and a complementary non-targetstrand.

Embodiment 37. The complex of embodiment 36, wherein the R-loopcomprises (i) an RNA-DNA hybrid comprising the PEgRNA and the targetstrand, and (ii) the complementary non-target strand.

Embodiment 38. The complex of embodiment 37, wherein the target or thecomplementary non-target strand is nicked to form a priming sequencehaving a free 3′ end.

Embodiment 39. The complex of embodiment 38, wherein the nick site isupstream of the PAM sequence on the target strand.

Embodiment 40. The complex of embodiment 38, wherein the nick site isupstream of the PAM sequence on the non-target strand.

Embodiment 41. The complex of embodiment 38, wherein the nick site-1,-2, -3, -4, -5, -6, -7, -8, or -9 relative to the 5′ end of the PAMsequence.

Embodiment 42. The complex of embodiment 22, wherein the single-strandDNA flap hybridizes to the endogenous DNA sequence adjacent to the nicksite, thereby installing the desired nucleotide change in the targetstrand.

Embodiment 43. The complex of embodiment 22, wherein the single-strandedDNA flap displaces the endogenous DNA sequence adjacent to the nick siteand which has a free 5′ end.

Embodiment 44. The complex of embodiment 22, wherein the endogenous DNAsequence having the 5′ end is excised by the cell.

Embodiment 45. The complex of embodiment 44, wherein the endogenous DNAsequence having the 5′ end is excised by a flap endonuclease.

Embodiment 46. The complex of embodiment 43, wherein cellular repair ofthe single-strand DNA flap incorporates the desired nucleotide change inthe non-target strand, thereby forming a desired product.

Embodiment 47. The complex of embodiment 46, wherein the desirednucleotide change is installed in an editing window that is betweenabout −4 to +10 of the PAM sequence, or between about −10 to +20 of thePAM sequence, or between about −20 to +40 of the PAM sequence, orbetween about −30 to +100 of the PAM sequence.

Embodiment 48. The complex of embodiment 47, wherein the desirednucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 nucleotides downstream of the nick site.

Embodiment 49. The complex of any one of the previous embodiments,wherein the fusion protein comprises the structureNH₂-[napDNAbp]-[domain comprising an RNA-dependent DNA polymeraseactivity]-COOH; or NH₂-[domain comprising an RNA-dependent DNApolymerase activity]-[napDNAbp]-COOH, wherein each instance of “]-[”indicates the presence of an optional linker sequence.

Embodiment 50. The complex of embodiment 49, wherein the linker sequencecomprises an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453,and 767-769.

Embodiment 51. The complex of embodiment 1, wherein the fusion proteinfurther comprises a linker that joins the napDNAbp and the domaincomprising an RNA-dependent DNA polymerase activity.

Embodiment 52. The complex of embodiment 51, wherein the linker sequencecomprises an amino acid sequence of SEQ ID NOs. 174 (1×SGGS), 446(2×SGGS), 3889 (3×SGGS), 171 (1×XTEN), 3968 (1×EAAAK), 3969 (2×EAAAK),and 3970 (3x EAAAK).

Group D. PE Method for Correcting Mutations

Embodiment 1. A method for installing a desired nucleotide change in adouble-stranded DNA sequence, the method comprising: contacting thedouble-stranded DNA sequence with a complex comprising a fusion proteinand a PEgRNA, wherein the fusion protein comprises a napDNAbp and apolymerase, and wherein the PEgRNA comprises a DNA synthesis templatecomprising the desired nucleotide change and a primer binding site;

-   -   thereby nicking the double-stranded DNA sequence, thereby        generating a free single-strand DNA having a 3′ end;    -   thereby hybridizing the 3′ end of the free single-strand DNA to        the primer binding site, thereby priming the polymerase;    -   thereby polymerizing a strand of DNA from the 3′ end hybridized        to the primer binding site, thereby generating a single-strand        DNA flap comprising the desired nucleotide change and which is        complementary to the DNA synthesis template;    -   thereby replacing an endogenous DNA strand adjacent the cut site        with the single-strand DNA flap, thereby installing the desired        nucleotide change in the double-stranded DNA sequence.

Embodiment 2. The method of embodiment 1, wherein replacing anendogenous DNA strand comprises: (i) hybridizing the single-strand DNAflap to the endogenous DNA strand adjacent the cut site to create asequence mismatch; (ii) excising the endogenous DNA strand; and (iii)repairing the mismatch to form the desired product comprising thedesired nucleotide change in both strands of DNA.

Embodiment 3. The method of embodiment 1, wherein the desired nucleotidechange is a single nucleotide substitution, a deletion, or an insertion.

Embodiment 4. The method of embodiment 3, wherein the single nucleotidesubstitution is a transition or a transversion.

Embodiment 5. The method of embodiment 1, wherein the desired nucleotidechange is (1) a G to T substitution, (2) a G to A substitution, (3) a Gto C substitution, (4) a T to G substitution, (5) a T to A substitution,(6) a T to C substitution, (7) a C to G substitution, (8) a C to Tsubstitution, (9) a C to A substitution, (10) an A to T substitution,(11) an A to G substitution, or (12) an A to C substitution.

Embodiment 6. The method of embodiment 1, wherein the desired nucleotidechange converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepairto an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:Abasepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) aT:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair,(8) a C:G basepair to a T:A basepair, a C:G basepair to an A:T basepair,(10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:Cbasepair, or (12) an A:T basepair to a C:G basepair.

Embodiment 7. The method of embodiment 1, wherein the desired nucleotidechange is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

Embodiment 8. The method of embodiment 1, wherein the desired nucleotidechange corrects a disease-associated gene.

Embodiment 9. The method of embodiment 8, wherein the disease-associatedgene is associated with a monogenetic disorder selected from the groupconsisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 AntitrypsinDeficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; MarfanSyndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease;Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a priondisease; and Tay-Sachs Disease.

Embodiment 10. The method of embodiment 8, wherein thedisease-associated gene is associated with a polygenic disorder selectedfrom the group consisting of: heart disease; high blood pressure;Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 11. The method of embodiment 1, wherein the napDNAbp is anuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease activeCas9.

Embodiment 12. The method of embodiment 1, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18.

Embodiment 13. The method of embodiment 1, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 14. The method of embodiment 1, wherein the polymerase is areverse transcriptase comprising any one of the amino acid sequences ofSEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235,454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 15. The method of embodiment 1, wherein the polymerase is areverse transcriptase comprising an amino acid sequence having at least80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acidsequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139,143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and766.

Embodiment 16. The method of embodiment 1, wherein the PEgRNA comprisesa nucleic acid extension arm at the 3′ or 5′ ends or at anintramolecular location in the guide RNA, wherein the extension armcomprises the DNA synthesis template sequence and the primer bindingsite.

Embodiment 17. The method of embodiment 16, wherein the extension arm isat least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides,at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides,at least 11 nucleotides, at least 12 nucleotides, at least 13nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, atleast 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides,at least 22 nucleotides, at least 23 nucleotides, at least 24nucleotides, or at least 25 nucleotides in length.

Embodiment 18. The method of embodiment 1, wherein the PEgRNA has anucleotide sequence selected from the group consisting of SEQ ID NOs:101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346,348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761,776-777.

Embodiment 19. A method for introducing one or more changes in thenucleotide sequence of a DNA molecule at a target locus, comprising:contacting the DNA molecule with a nucleic acid programmable DNA bindingprotein (napDNAbp) and a PEgRNA which targets the napDNAbp to the targetlocus, wherein the PEgRNA comprises a reverse transcriptase (RT)template sequence comprising at least one desired nucleotide change anda primer binding site;

-   -   thereby forming an exposed 3′ end in a DNA strand at the target        locus;    -   thereby hybridizing the exposed 3′ end to the primer binding        site to prime reverse transcription;    -   thereby synthesizing a single strand DNA flap comprising the at        least one desired nucleotide change based on the RT template        sequence by reverse transcriptase;    -   thereby incorporating the at least one desired nucleotide change        into the corresponding endogenous DNA, thereby introducing one        or more changes in the nucleotide sequence of the DNA molecule        at the target locus.

Embodiment 20. The method of embodiment 19, wherein the one or morechanges in the nucleotide sequence comprises a transition.

Embodiment 21. The method of embodiment 19, wherein the transition isselected from the group consisting of: (a) T to C; (b) A to G; (c) C toT; and (d) G to A.

Embodiment 22. The method of embodiment 19, wherein the one or morechanges in the nucleotide sequence comprises a transversion.

Embodiment 23. The method of embodiment 22, wherein the transversion isselected from the group consisting of: (a) T to A; (b) T to G; (c) C toG; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.

Embodiment 24. The method of embodiment 19, wherein the one or morechanges in the nucleotide sequence comprises changing (1) a G:C basepairto a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:Cbasepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) aT:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair,(7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:Abasepair, (9) a C:G basepair to an A:T basepair, (9) an A:T basepair toa T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:Tbasepair to a C:G basepair.

Embodiment 25. The method of embodiment 19, wherein the one or morechanges in the nucleotide sequence comprises an insertion or deletion of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25 nucleotides.

Embodiment 26. The method of embodiment 19, wherein the one or morechanges in the nucleotide sequence comprises a correction to adisease-associated gene.

Embodiment 27. The method of embodiment 26, wherein thedisease-associated gene is associated with a monogenetic disorderselected from the group consisting of: Adenosine Deaminase (ADA)Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; DuchenneMuscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease;Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1;Pachyonychia Congenita; Phenylkeotnuria; Severe CombinedImmunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; atrinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.

Embodiment 28. The method of embodiment 26, wherein thedisease-associated gene is associated with a polygenic disorder selectedfrom the group consisting of: heart disease; high blood pressure;Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 29. The method of embodiment 19, wherein the napDNAbp is anuclease active Cas9 or variant thereof.

Embodiment 30. The method of embodiment 19, wherein the napDNAbp is anuclease inactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variantthereof.

Embodiment 31. The method of embodiment 19, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18.

Embodiment 32. The method of embodiment 19, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 33. The method of embodiment 19, wherein the reversetranscriptase is introduced in trans.

Embodiment 34. The method of embodiment 19, wherein the napDNAbpcomprises a fusion to a reverse transcriptase.

Embodiment 35. The method of embodiment 19, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 36. The method of embodiment 19, wherein the reversetranscriptase comprises an amino acid sequence having at least 80%, 85%,90%, 95%, 98%, or 99% sequence identity with the amino acid sequence ofany one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149,154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 37. The method of embodiment 19, wherein the step of formingan exposed 3′ end in the DNA strand at the target locus comprisesnicking the DNA strand with a nuclease.

Embodiment 38. The method of embodiment 37, wherein the nuclease is thenapDNAbp, is provided as a fusion domain of napDNAbp, or is provided intrans.

Embodiment 39. The method of embodiment 19, wherein the step of formingan exposed 3′ end in the DNA strand at the target locus comprisescontacting the DNA strand with a chemical agent.

Embodiment 40. The method of embodiment 19, wherein the step of formingan exposed 3′ end in the DNA strand at the target locus comprisesintroducing a replication error.

Embodiment 41. The method of embodiment 19, wherein the step ofcontacting the DNA molecule with the napDNAbp and the guide RNA forms anR-loop.

Embodiment 42. The method of embodiment 41, wherein the DNA strand inwhich the exposed 3′ end is formed is in the R-loop.

Embodiment 43. The method of embodiment 19, wherein the PEgRNA comprisesan extension arm that comprises the reverse transcriptase (RT) templatesequence and the primer binding site.

Embodiment 44. The method of embodiment 43, wherein the extension arm isat the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at anintramolecular position in the guide RNA.

Embodiment 45. The method of embodiment 19, wherein the PEgRNA furthercomprises at least one additional structure selected from the groupconsisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer,or an RNA-protein recruitment domain.

Embodiment 46. The method of embodiment 19, wherein the PEgRNA furthercomprises a homology arm.

Embodiment 47. The method of embodiment 19, wherein the RT templatesequence is homologous to the corresponding endogenous DNA.

Embodiment 48. A method for introducing one or more changes in thenucleotide sequence of a DNA molecule at a target locus by target-primedreverse transcription, the method comprising: (a) contacting the DNAmolecule at the target locus with a (i) fusion protein comprising anucleic acid programmable DNA binding protein (napDNAbp) and a reversetranscriptase and (ii) a guide RNA comprising an RT template comprisinga desired nucleotide change;

-   -   thereby conducting target-primed reverse transcription of the RT        template to generate a single strand DNA comprising the desired        nucleotide change;    -   thereby incorporating the desired nucleotide change into the DNA        molecule at the target locus through a DNA repair and/or        replication process.

Embodiment 49. The method of embodiment 48, wherein the RT template islocated at the 3′ end of the guide RNA, the 5′ end of the guide RNA, orat an intramolecular location in the guide RNA.

Embodiment 50. The method of embodiment 48, wherein the desirednucleotide change comprises a transition, a transversion, an insertion,or a deletion, or any combination thereof.

Embodiment 51. The method of embodiment 48, wherein the desirednucleotide change comprises a transition selected from the groupconsisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.

Embodiment 52. The method of embodiment 48, wherein the desirednucleotide change comprises a transversion selected from the groupconsisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A toT; (f) A to C; (g) G to C; and (h) G to T.

Embodiment 53. The method of embodiment 48, wherein the desirednucleotide change comprises changing (1) a G:C basepair to a T:Abasepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair toC:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepairto an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:Gbasepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) aC:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair,(11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:Gbasepair.

Embodiment 54. A method for replacing a trinucleotide repeat expansionmutation in a target DNA molecule with a healthy sequence comprising ahealthy number of repeat trinucleotides, the method comprising: (a)contacting the DNA molecule at the target locus with a (i) fusionprotein comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a polymerase and (ii) a PEgRNA comprising DNA synthesistemplate comprising the replacement sequence and a primer binding site;(b) conducting prime editing to generate a single strand DNA comprisingthe replacement sequence; and (c) incorporating the single strand DNAinto the DNA molecule at the target locus through a DNA repair and/orreplication process.

Embodiment 55. The method of embodiment 54, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 56. The method of embodiment 54, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 57. The method of embodiment 54, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 58. The method of embodiment 54, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 59. The method of embodiment 54, wherein the step of (b)conducting prime editing comprises generating a 3′ end primer bindingsequence at the target locus that is capable of priming polymerase byannealing to the primer binding site on the guide RNA.

Embodiment 60. The method of embodiment 54, wherein the trinucleotiderepeat expansion mutation is associated with Huntington's Disease,Fragile X syndrome, or Friedreich's ataxia.

Embodiment 61. The method of embodiment 54, wherein the trinucleotiderepeat expansion mutation comprises a repeating unit of CAG triplets.

Embodiment 62. The method of embodiment 54, wherein the trinucleotiderepeat expansion mutation comprises a repeating unit of GAA triplets.

Embodiment 63. A method for introducing one or more changes in thenucleotide sequence of a DNA molecule at a target locus by target-primedreverse transcription, the method comprising: (a) contacting the DNAmolecule at the target locus with a (i) fusion protein comprising anucleic acid programmable DNA binding protein (napDNAbp) and a reversetranscriptase and (ii) a guide RNA comprising an RT template comprisinga desired nucleotide change;

-   -   thereby conducting target-primed reverse transcription of the RT        template to generate a single strand DNA comprising the desired        nucleotide change;    -   thereby incorporating the desired nucleotide change into the DNA        molecule at the target locus through a DNA repair and/or        replication process.

Embodiment 64. The method of embodiment 63, wherein the RT template islocated at the 3′ end of the guide RNA, the 5′ end of the guide RNA, orat an intramolecular location in the guide RNA.

Embodiment 65. The method of embodiment 63, wherein the desirednucleotide change comprises a transition, a transversion, an insertion,or a deletion, or any combination thereof.

Embodiment 66. The method of embodiment 63, wherein the desirednucleotide change comprises a transition selected from the groupconsisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.

Embodiment 67. The method of embodiment 63, wherein the desirednucleotide change comprises a transversion selected from the groupconsisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A toT; (f) A to C; (g) G to C; and (h) G to T.

Embodiment 68. The method of embodiment 63, wherein the desirednucleotide change comprises changing (1) a G:C basepair to a T:Abasepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair toC:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepairto an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:Gbasepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) aC:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair,(11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:Gbasepair.

Embodiment 69. A method of preventing or halting the progression of aprion disease by installing on or more protective mutations into PRNPencoded by a target nucleotide sequence by prime editing, the methodcomprising: (a) contacting the target nucleotide sequence with a (i)prime editor comprising a nucleic acid programmable DNA binding protein(napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edittemplate encoding the functional moiety;

-   -   thereby polymerizing a single strand DNA sequence encoding the        protective mutation;    -   thereby incorporating the single strand DNA sequence in place of        a corresponding endogenous strand at the target nucleotide        sequence through a DNA repair and/or replication process;    -   wherein the method produces a recombinant target nucleotide        sequence that encodes a PRNP comprising a protective mutation        and which is resistant to misfolding.

Embodiment 70. The method of embodiment 69, wherein the prion disease isa human prion disease.

Embodiment 71. The method of embodiment 69, wherein the prion disease isan animal prion disease.

Embodiment 72. The method of embodiment 69, wherein the prion disease isCreutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease(vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal FamilialInsomnia, or Kuru.

Embodiment 73. The method of embodiment 69, wherein the prion disease isBovine Spongiform Encephalopathy (BSE or “mad cow disease”), ChronicWasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy,Feline Spongiform Encephalopathy, and Ungulate SpongiformEncephalopathy.

Embodiment 74. The method of embodiment 69, wherein the wildtype PRNPamino acid sequence is SEQ ID NOs: 291-292.

Embodiment 75. The method of embodiment 69, wherein the method resultsin a modified PRNP amino acid sequence selected from the groupconsisting of SEQ ID NOs: 293-309, 311-323, wherein said modified PRNPprotein is resistant to misfolding.

Embodiment 76. The method of embodiment 69, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 77. The method of embodiment 69, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 78. The method of embodiment 69, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 79. The method of embodiment 69, wherein the PEgRNA comprisesSEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 80. A method of treating CDKL5 Deficiency Disorder bycorrecting a mutation in the cyclin-dependent kinase-like 5 gene (CDKL5)in a target nucleotide sequence by prime editing, the method comprising:(a) contacting the target nucleotide sequence with a (i) prime editorcomprising a nucleic acid programmable DNA binding protein (napDNAbp)and a polymerase and (ii) a PEgRNA comprising an edit template thatcorrects the mutation in CDKL5;

-   -   thereby polymerizing a single strand DNA sequence encoding the        edit;    -   thereby incorporating the single strand DNA sequence in place of        a corresponding endogenous strand at the target nucleotide        sequence through a DNA repair and/or replication process;    -   wherein the method produces a recombinant target nucleotide        sequence that encodes a repaired CDKL5 gene.

Embodiment 81. The method of embodiment D80, wherein the mutation inCDKL5 is 1412delA.

Group E. PE Methods for Modifying Protein Structure/Function and/orMutagenesis

Embodiment 1. A method for mutagenizing a DNA molecule at a target locusby prime editing, the method comprising: (a) contacting the DNA moleculeat the target locus with a (i) fusion protein comprising a nucleic acidprogrammable DNA binding protein (napDNAbp) and an error-prone polymer(e.g., error-prone reverse transcriptase), and (ii) a guide RNAcomprising an edit template comprising a desired nucleotide change;thereby polymerizing a single stranded DNA templated from the edittemplate; and incorporating the single stranded DNA into the DNAmolecule at the target locus through a DNA repair and/or replicationprocess.

Embodiment 2. The method of any prior embodiment, wherein the fusionprotein comprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 3. The method of any prior embodiment, wherein the napDNAbpis a Cas9 nickase (nCas9).

Embodiment 4. The method of embodiment 1, wherein the napDNAbp comprisesthe amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147,153, 157, 445, 460, 467, and 482-487.

Embodiment 5. The method of embodiment 1, wherein the guide RNAcomprises SEQ ID NO: 222.

Embodiment 6. A method of installing an immunoepitope in a protein ofinterest encoded by a target nucleotide sequence by prime editing, themethod comprising: (a) contacting the target nucleotide sequence with a(i) prime editor comprising a nucleic acid programmable DNA bindingprotein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edittemplate encoding the functional moiety;

-   -   thereby polymerizing a single strand DNA sequence encoding the        immunoepitope;    -   thereby incorporating the single strand DNA sequence in place of        a corresponding endogenous strand at the target nucleotide        sequence through a DNA repair and/or replication process;    -   wherein the method produces a recombinant target nucleotide        sequence that encodes a fusion protein comprising the protein of        interest and the immunoepitope.

Embodiment 7. The method of embodiment 6, wherein the immunoepitope isselected from the group consisting of: tetanus toxoid (SEQ ID NO: 396);diphtheria toxin mutant CRM197 (SEQ ID NO: 630); mumps immunoepitope 1(SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumpsimmunoepitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406);hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1(SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes towardclass I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA(SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO:420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422);hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO:424); and neuraminidase epitope H5N1-bound class I and class II HLA (SEQID NO: 426).

Embodiment 8. The method of embodiment 6, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 9. The method of embodiment 6, wherein the napDNAbp is a Cas9nickase (nCas9).

Embodiment 10. The method of embodiment 6, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 11. The method of embodiment 6, wherein the PEgRNA comprisesSEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 12. A method of installing a small molecule dimerizationdomain in a protein of interest encoded by a target nucleotide sequenceby prime editing, the method comprising: (a) contacting the targetnucleotide sequence with a (i) prime editor comprising a nucleic acidprogrammable DNA binding protein (napDNAbp) and a polymerase, and (ii) aPEgRNA comprising an edit template encoding the small moleculedimerization domain;

-   -   thereby polymerizing a single strand DNA sequence encoding the        immunoepitope;    -   thereby incorporating the single strand DNA sequence in place of        a corresponding endogenous strand at the target nucleotide        sequence through a DNA repair and/or replication process;    -   wherein the method produces a modified target nucleotide        sequence that encodes a fusion protein comprising the protein of        interest and the small molecule dimerization domain.

Embodiment 13. The method of embodiment 12 further comprising conductingthe method on a second protein of interest.

Embodiment 14. The method of embodiment 13, wherein the first protein ofinterest and the second protein of interest dimerize in the presence ofa small molecule that binds to the dimerization domain on each of saidproteins.

Embodiment 15. The method of embodiment 12, wherein the small moleculebinding domain is FKBP12 of SEQ ID NO: 488.

Embodiment 16. The method of embodiment 12, wherein the small moleculebinding domain is FKBP12-F36V of SEQ ID NO: 489.

Embodiment 17. The method of embodiment 12, wherein the small moleculebinding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.

Embodiment 18. The method of embodiment 12, wherein the small moleculeis a dimer of a small molecule as described herein.

Embodiment 19. The method of embodiment 12, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 20. The method of embodiment 12, wherein the napDNAbp is aCas9 nickase (nCas9).

Embodiment 21. The method of embodiment 12, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137,141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 22. The method of embodiment 12, wherein the PEgRNA comprisesSEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 23. A method of installing a peptide tag or epitope onto aprotein using prime editing, comprising: contacting a target nucleotidesequence encoding the protein with a prime editor construct configuredto insert therein a second nucleotide sequence encoding the peptide tagto result in a recombinant nucleotide sequence, such that the peptidetag and the protein are expressed from the recombinant nucleotidesequence as a fusion protein.

Embodiment 24. The method of embodiment 23, wherein the peptide tag isused for purification and/or detection of the protein.

Embodiment 25. The method of embodiment 23, wherein the peptide tag is apoly-histidine (e.g., HHHHHH SEQ ID NOs: 252-262), FLAG (e.g., DYKDDDDK)(SEQ ID NO: 2), V5 (e.g., GKPIPNPLLGLDST) (SEQ ID NO: 3), GCN4, HA(e.g., YPYDVPDYA) (SEQ ID NO: 5), Myc (e.g. EQKLISEED) (SEQ ID NO: 6),or GST.

Embodiment 26. The method of embodiment 23, wherein the peptide tag hasan amino acid sequence selected from the group consisting of SEQ ID NO:1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622.

Embodiment 27. The method of embodiment 23, wherein the peptide tag isfused to the protein by a linker.

Embodiment 28. The method of embodiment 23, wherein the fusion proteinhas the following structure: [protein]-[peptide tag] or [peptidetag]-[protein], wherein “]-[” represents an optional linker.

Embodiment 29. The method of embodiment 23, wherein the linker has anamino acid sequence of SEQ ID NO: 127, 165-176, 446,453, and 767-769.

Embodiment 30. The method of embodiment 23, wherein the prime editorconstruct comprises a PEgRNA comprising the nucleotide sequence of SEQID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777.

Embodiment 31. The method of embodiment 23, wherein the PEgRNA comprisesa spacer, a gRNA core, and an extension arm, wherein the spacer iscomplementary to the target nucleotide sequence and the extension armcomprises a reverse transcriptase template that encodes the peptide tag.

Embodiment 32. The method of embodiment 23, wherein the PEgRNA comprisesa spacer, a gRNA core, and an extension arm, wherein the spacer iscomplementary to the target nucleotide sequence and the extension armcomprises a reverse transcriptase template that encodes the peptide tag.

Embodiment 33. A method of installing or deleting a functional moiety ina protein of interest encoded by a target nucleotide sequence by primeediting, the method comprising: (a) contacting the target nucleotidesequence with a (i) prime editor comprising a nucleic acid programmableDNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNAcomprising an edit template encoding the functional moiety or deletionof same; (b) polymerizing a single strand DNA sequence encoding thefunctional moiety or deletion of same; and (c) incorporating the singlestrand DNA sequence in place of a corresponding endogenous strand at thetarget nucleotide sequence through a DNA repair and/or replicationprocess, wherein the method produces a recombinant target nucleotidesequence that encodes a modified protein comprising the protein ofinterest and the functional moiety or the removal of same, wherein thefunctional moiety alters a modification state or localization state ofthe protein.

Embodiment 34. The method of embodiment 33, wherein functional moietyalters the phosphorylation, ubiquitylation, glycosylation, lipidation,hydroxylation, methylation, acetylation, crotonylation, or SUMOylationstate of the protein of interest.

Group F. PE Delivery Methods and Compositions

Embodiment 1. A polypeptide comprising an N-terminal half or aC-terminal half of a prime editor fusion protein.

Embodiment 2. The polypeptide of embodiment 1, wherein the prime editorfusion protein comprises a nucleic acid programmable DNA binding protein(napDNAbp) domain and a polymerase domain.

Embodiment 3. The polypeptide of embodiment 1, wherein the prime editorfusion protein is capable of carrying out prime editing in the presenceof a prime editing guide RNA (PEgRNA).

Embodiment 4. The polypeptide of embodiment 2, wherein the napDNAbp is aCas9 protein or variant thereof.

Embodiment 5. The polypeptide of embodiment 2, wherein the napDNAbp is anuclease with nickase activity.

Embodiment 6. The polypeptide of embodiment 2, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

Embodiment 7. The polypeptide of embodiment 2, wherein the napDNAbp isselected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a,Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickaseactivity.

Embodiment 8. The polypeptide of embodiment 1, wherein the polypeptideis formed by splitting a prime editor fusion protein at a split site.

Embodiment 9. The polypeptide of embodiment 8, wherein the split site isa peptide bond in the napDNAbp domain.

Embodiment 10. The polypeptide of embodiment 8, wherein the split siteis a peptide bond in the polymerase domain.

Embodiment 11. The polypeptide of embodiment 8, wherein the split siteis a peptide bond in a linker between the napDNAbp domain and thepolymerase domain.

Embodiment 12. The polypeptide of embodiment 9, wherein the split siteis in the peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12,12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33,33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residuesbetween residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350,350-400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000,1000-1100, 1100-1200, 1200-1300, or 1300-1368 of SEQ ID NO: 18(canonical SpCas9), or between any two equivalent amino acid residues ofan SpCas9 homolog or equivalent of SEQ ID NO: 18.

Embodiment 13. The polypeptide of embodiment 9, wherein the split siteis in the peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12,12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33,33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residuesbetween residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350,350-400, 400-450, 450-500, 500-600, or 600-667 of SEQ ID NO: 89(canonical reverse transcriptase, M-MLV RT), or between any twoequivalent amino acid residues of a reverse transcriptase homolog orequivalent of SEQ ID NO: 89.

Embodiment 14. The polypeptide of embodiment 1, wherein the polypeptideis an N-terminal half of a prime editor fusion protein.

Embodiment 15. The polypeptide of embodiment 1, wherein the polypeptideis a C-terminal half of a prime editor fusion protein.

Embodiment 16. A nucleotide sequence encoding a polypeptide of any ofembodiments 1-15 and optionally a PEgRNA.

Embodiment 17. A virus genome comprising a nucleotide sequence encodinga polypeptide of any of embodiments 1-15 and optionally a PEgRNA.

Embodiment 18. The virus genome of embodiment 17, wherein the nucleotidesequence further comprises a promoter sequence suitable for expressingthe polypeptide of any of embodiments 1-15.

Embodiment 19. The virus genome of embodiment 17, wherein the nucleotidesequence further comprises a sequence encoding a PEgRNA.

Embodiment 20. A virus particle comprising a genome comprising anucleotide sequence encoding a polypeptide of any of embodiments 1-15and optionally a PEgRNA.

Embodiment 21. The virus particle of embodiment 20, wherein the virusparticle is an adenovirus particle, an adeno-associated virus particle,or a lentivirus particle.

Embodiment 22. The virus particle of embodiment 20, wherein thepolypeptide encoded by the genome is an N terminal half of a primeeditor fusion protein.

Embodiment 23. The virus particle of embodiment 20, wherein thepolypeptide encoded by the genome is a C terminal half of a prime editorfusion protein.

Embodiment 24. A pharmaceutical composition comprising a virus particleof any of embodiments 20-23 and a pharmaceutical excipient.

Embodiment 25. A pharmaceutical composition comprising a virus particleof embodiment 22 (encoding the N terminal half) and a pharmaceuticalexcipient.

Embodiment 26. A pharmaceutical composition comprising a virus particleof embodiment 23 (encoding the C terminal half) and a pharmaceuticalexcipient.

Embodiment 27. A ribonucleoprotein (RNP) complex comprising a nucleotidesequence encoding a polypeptide of any of embodiments 1-15 andoptionally a PEgRNA.

Embodiment 28. The ribonucleoprotein (RNP) complex of embodiment 27,wherein the polypeptide encoded by the genome is an N-terminal half of aprime editor fusion protein.

Embodiment 29. The ribonucleoprotein (RNP) complex of embodiment 27,wherein the polypeptide encoded by the genome is a C-terminal half of aprime editor fusion protein.

Embodiment 30. A pharmaceutical composition comprising aribonucleoprotein (RNP) complex of any of embodiments 27-29 and apharmaceutical excipient.

Embodiment 31. A pharmaceutical composition comprising theribonucleoprotein (RNP) complex of embodiment 28 (encoding theN-terminal half) and a pharmaceutical excipient.

Embodiment 32. A pharmaceutical composition comprising theribonucleoprotein (RNP) complex of embodiment 29 (encoding theC-terminal half) and a pharmaceutical excipient.

Embodiment 33. A pharmaceutical composition comprising a first AAVparticle and a second AAV particle, wherein the first AAV vectorexpresses an N-terminal half of a prime editor fusion protein and thesecond AAV vector expresses a C-terminal half of a prime editor fusionprotein, wherein the N-terminal half and the C-terminal half arecombined within the cell to reconstitute the prime editor.

Embodiment 34. The pharmaceutical composition of embodiment 33, whereinthe first or second AAV particle also expresses a PEgRNA that targetsthe reconstituted prime editor to a target DNA site.

Embodiment 35. The pharmaceutical composition of embodiment 33, whereinthe prime editor fusion protein comprises a nucleic acid programmableDNA binding protein (napDNAbp) domain and a polymerase domain.

Embodiment 36. The pharmaceutical composition of embodiment 33, whereinthe prime editor fusion protein is capable of carrying out prime editingin the presence of a prime editing guide RNA (PEgRNA).

Embodiment 37. The pharmaceutical composition of embodiment 35, whereinthe napDNAbp is a Cas9 protein or variant thereof.

Embodiment 38. The pharmaceutical composition of embodiment 35, whereinthe napDNAbp is a nuclease with a nickase activity.

Embodiment 39. The pharmaceutical composition of embodiment 35, whereinthe napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9(dCas9), or a Cas9 nickase (nCas9).

Embodiment 40. The pharmaceutical composition of embodiment 35, whereinthe napDNAbp is selected from the group consisting of: Cas9, Cas12e,Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionallyhas a nickase activity.

Embodiment 41. The pharmaceutical composition of embodiment 33, whereinN-terminal and C-terminal halves are formed by splitting the primeeditor fusion protein at a split site.

Embodiment 42. The pharmaceutical composition of embodiment 41, whereinthe split site is a peptide bond in a napDNAbp domain.

Embodiment 43. The pharmaceutical composition of embodiment 41, whereinthe split site is a peptide bond in a polymerase domain.

Embodiment 44. The pharmaceutical composition of embodiment 41, whereinthe split site is a peptide bond in a linker.

Embodiment 45. The pharmaceutical composition of embodiment 41, whereinthe split site is in the peptide bond between residues 1 and 2, 2 and 3,3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and25, 25 and 26, 26 and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, orbetween any two residues between residues 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or1300-1368 of SEQ ID NO: 18 (canonical SpCas9), or between any twoequivalent amino acid residues of an SpCas9 homolog or equivalent of SEQID NO: 18.

Embodiment 46. The pharmaceutical composition of embodiment 41, whereinthe split site is in the peptide bond between residues 1 and 2, 2 and 3,3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and25, 25 and 26, 26 and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, orbetween any two residues between residues 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, or600-667 of SEQ ID NO: 89 (canonical reverse transcriptase, M-MLV RT), orbetween any two equivalent amino acid residues of a reversetranscriptase homolog or equivalent of SEQ ID NO: 89.

Embodiment 47. The pharmaceutical composition of embodiment 33, whereinthe N-terminal half of the prime editor fusion protein has an amino acidsequence encoding the N-terminal prime editor fusion proteins asdescribed herein.

Embodiment 48. The pharmaceutical composition of embodiment 33, whereinthe C-terminal half of the prime editor fusion protein has an amino acidsequence encoding the N-terminal prime editor fusion proteins asdescribed herein.

Embodiment 49. A method of delivering a prime editor fusion protein to acell comprising transfecting the cell with a first AAV particle and asecond AAV particle, wherein the first AAV vector expresses anN-terminal half of a prime editor fusion protein and the second AAVvector expresses a C-terminal half of a prime editor fusion protein,wherein the N-terminal half and the C-terminal half are combined withinthe cell to reconstitute the prime editor fusion protein.

Embodiment 50. The method of embodiment 49, wherein the first or secondAAV particle also expresses a PEgRNA that targets the reconstitutedprime editor to a target DNA site.

Embodiment 51. The method of embodiment 49, wherein the prime editorfusion protein comprises a nucleic acid programmable DNA binding protein(napDNAbp) domain and a polymerase domain.

Embodiment 52. The method of embodiment 49, wherein the prime editorfusion protein is capable of carrying out prime editing in the presenceof an prime editing guide RNA (PEgRNA).

Embodiment 53. The method of embodiment 51, wherein the napDNAbp is aCas9 protein or variant thereof.

Embodiment 54. The method of embodiment 53, wherein the napDNAbp is anuclease with nickase activity.

Embodiment 55. The method of embodiment 53, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

Embodiment 56. The method of embodiment 53, wherein the napDNAbp isselected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a,Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickaseactivity.

Embodiment 57. The method of embodiment 49, wherein N-terminal andC-terminal halves are formed by splitting the prime editor fusionprotein at a split site.

Embodiment 58. The method of embodiment 57, wherein the split site is apeptide bond in a napDNAbp domain.

Embodiment 59. The method of embodiment 57, wherein the split site is apeptide bond in a polymerase domain.

Embodiment 60. The method of embodiment 57, wherein the split site is apeptide bond in a linker.

Embodiment 61. The method of embodiment 57, wherein the split site is inthe peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residuesbetween residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350,350-400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000,1000-1100, 1100-1200, 1200-1300, or 1300-1368 of SEQ ID NO: 18(canonical SpCas9), or between any two equivalent amino acid residues ofan SpCas9 homolog or equivalent of SEQ ID NO: 18.

Embodiment 62. The method of embodiment 57, wherein the split site is inthe peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residuesbetween residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350,350-400, 400-450, 450-500, 500-600, or 600-667 of SEQ ID NO: 89(canonical reverse transcriptase, M-MLV RT), or between any twoequivalent amino acid residues of a reverse transcriptase homolog orequivalent of SEQ ID NO: 89.

Embodiment 63. The method of embodiment 49, wherein the N-terminal halfof the prime editor fusion protein has an amino acid sequence encodingthe N-terminal prime editor fusion proteins as described herein.

Embodiment 64. The method of embodiment 49, wherein the C-terminal halfof the prime editor fusion protein has an amino acid sequence encodingthe C-terminal prime editor fusion proteins as described herein.

Embodiment 65. The method of embodiment 49, wherein the first AAVparticle comprises a recombinant AAV genome comprising a nucleotidesequence which encodes the first prime editor component.

Embodiment 66. The method of embodiment 49, wherein the second AAVparticle comprises a recombinant AAV genome comprising a nucleotidesequence which encodes the second prime editor component.

Embodiment 67. The method of embodiment 49, wherein the transfectingstep is conducted in vivo.

Embodiment 68. The method of embodiment 49, wherein the transfectingstep is conducted ex vivo.

Embodiment 69. The method of embodiment 50, wherein the target DNA siteis a disease-associated gene.

Embodiment 70. The method of embodiment 69, wherein thedisease-associated gene is associated with a monogenetic disorderselected from the group consisting of: Adenosine Deaminase (ADA)Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; DuchenneMuscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease;Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1;Pachyonychia Congenita; Phenylkeotnuria; Severe CombinedImmunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; atrinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.

Embodiment 71. The method of embodiment 69, wherein thedisease-associated gene is associated with a polygenic disorder selectedfrom the group consisting of: heart disease; high blood pressure;Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 72. The method of embodiment 51, wherein the programmable DNAbinding protein (napDNAbp) domain.

Embodiment 73. The method of embodiment 51, wherein the polymerasedomain is a reverse transcriptase.

Embodiment 74. The method of embodiment 73, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 75. The method of embodiment 73, wherein the reversetranscriptase comprises an amino acid sequence having at least 80%, 85%,90%, 95%, 98%, or 99% sequence identity with the amino acid sequence ofany one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149,154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 76. The method of embodiment 73, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18.

Embodiment 77. The method of embodiment 73, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487. Embodiment 78. The polypeptide of embodiment 8, wherein thesplit site is between 1023 and 1024 of SEQ ID NO: 18, or at acorresponding position in an amino acid sequence having at least 85%, atleast 90%, at least 95%, at least 99%, or at least 99.5% sequenceidentity with SEQ ID NO: 18.

Group G. PE Methods for Modifying RNA Structure/Function

Embodiment 1. A method of installing a ribonucleotide motif or tag in anRNA of interest encoded by a target nucleotide sequence by primeediting, the method comprising: (a) contacting the target nucleotidesequence with a (i) prime editor comprising a nucleic acid programmableDNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNAcomprising an edit template encoding the ribonucleotide motif or tag;thereby polymerizing a single strand DNA sequence encoding theribonucleotide motif or tag; and incorporating the single strand DNAsequence in place of a corresponding endogenous strand at the targetnucleotide sequence through a DNA repair and/or replication process,wherein the method produces a target nucleotide sequence that encodes amodified RNA of interest comprising the ribonucleotide motif or tag.

Embodiment 2. The method of embodiment 1, wherein ribonucleotide motifor tag is a detection moiety.

Embodiment 3. The method of embodiment 1, wherein the ribonucleotidemotif or tag affects the expression level of the RNA of interest.

Embodiment 4. The method of embodiment 1, wherein the ribonucleotidemotif or tag affects the transport or subcellular location of the RNA ofinterest.

Embodiment 5. The method of embodiment 1, wherein the ribonucleotidemotif or tag is selected from the group consisting of SV40 type 1, SV40type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PANENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer,spinach aptamer, mango aptamer, HDV ribozyme, and m6A.

Embodiment 6. The method of embodiment 1, wherein the PEgRNA comprisesSEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,499-505, 735-761, 776-777 (see Table).

Embodiment 7. The method of embodiment 1, wherein the fusion proteincomprises the amino acid sequence of PE1, PE2, or PE3.

Embodiment 8. The method of embodiment 1, wherein the napDNAbp is a Cas9nickase (nCas9).

Embodiment 9. The method of embodiment 1, wherein the napDNAbp comprisesthe amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147,153, 157, 445, 460, 467, and 482-487.

Group H. PE Methods for Making Gene Libraries

Embodiment 1. A method of constructing a programmed mutant gene libraryby prime editing, the method comprising:

-   -   (a) contacting a library of target nucleotide sequences each        comprising one or more target genetic loci with a (i) prime        editor comprising a nucleic acid programmable DNA binding        protein (napDNAbp) and a polymerase, and (ii) a PEgRNA        comprising an edit template comprising a sequence having at        least one genetic change relative to the one or more target        genetic loci;    -   thereby polymerizing a single strand DNA sequence templated by        the edit template; and    -   incorporating the single strand DNA sequence in place of the one        or more target genetic loci through a DNA repair and/or        replication process, thereby incorporating the at least one        genetic change into the target genetic loci of the target        nucleotide sequences of said library.

Embodiment 2. The method of embodiment 1, wherein the library is aplasmid library.

Embodiment 3. The method of embodiment 1, wherein the library is a phagelibrary.

Embodiment 4. The method of embodiment 1, wherein the one or more targetgenetic loci comprise a region encoding a protein.

Embodiment 5. The method of embodiment 1, wherein the one or more targetgenetic loci comprise a region encoding a secondary structure motif of aprotein.

Embodiment 6. The method of embodiment 5, wherein the secondarystructure motif is an alpha helix.

Embodiment 7. The method of embodiment 5, wherein the secondarystructure motif is a beta sheet.

Embodiment 8. The method of embodiment 1, wherein the napDNAbp is a Cas9protein or variant thereof.

Embodiment 9. The method of embodiment 1, wherein the napDNAbp is anuclease with a nickase activity.

Embodiment 10. The method of embodiment 1, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

Embodiment 11. The method of embodiment 1, wherein the napDNAbpcomprises an amino acid sequence of SEQ ID NO: 18.

Embodiment 12. The method of embodiment 1, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 13. The method of embodiment 1, wherein the polymerase domainis a reverse transcriptase.

Embodiment 14. The method of embodiment 13, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 15. The method of embodiment 14, wherein the reversetranscriptase comprises an amino acid sequence having at least 80%, 85%,90%, 95%, 98%, or 99% sequence identity with the amino acid sequence ofany one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149,154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 16. The method of embodiment 1, wherein the at least onegenetic change in the edit template is an insertion.

Embodiment 17. The method of embodiment 1, wherein the at least onegenetic change in the edit template is a deletion.

Embodiment 18. The method of embodiment 1, wherein the at least onegenetic change in the edit template is substitution.

Embodiment 19. The method of embodiment 1, wherein the at least onegenetic change is an insertion of one or more codons.

Embodiment 20. The method of embodiment 1, wherein the at least onegenetic change is a deletion of one or more codons.

Embodiment 21. The method of embodiment 1, wherein the at least onegenetic change is the insertion of a stop codon.

Embodiment 22. The method of embodiment 1, wherein the at least onegenetic change is the conversion of a non-stop codon to a stop codon.

Embodiment 23. The method of embodiment 1, wherein the method is conductsimultaneously at least 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or10, or between 10-100, or between 100-200, or between 200-300, orbetween 300-400, or between 400-500 target genetic loci in each of thetarget nucleotide sequences of the library.

Embodiment 24. The method of embodiment 1, wherein the method isconducted during PACE or PANCE evolution, and wherein each instance ofincorporating the at least one genetic change into the target geneticloci of the target nucleotide sequences of said library also installs anew target sequence.

Group I. PE Methods for Off-Target Detection

Embodiment 1. A method of evaluating off-target editing by a primeeditor, the method comprising:

-   -   (a) contacting a target nucleotide sequence having an edit site        with a (i) prime editor fusion protein comprising a nucleic acid        programmable DNA binding protein (napDNAbp) and a polymerase        and (ii) a PEgRNA comprising a DNA synthesis template that        encodes a detectable sequence;    -   wherein the PEgRNA complexes with the fusion protein and guides        said fusion protein to the edit site and, if present, to one or        more off-target sites;    -   and wherein the prime editor fusion protein installs the        detectable sequence at the edit site, and, if present, at the        one or more off-target sites;    -   (b) determining the nucleotide sequence of the edit site and the        one or more off-target sites.

Embodiment 2. The method of embodiment 1, wherein the target nucleotidesequence is a genome.

Embodiment 3. The method of embodiment 1, wherein the step of contactingis in vitro.

Embodiment 4. The method of embodiment 1, wherein the step of contactingis in vivo.

Embodiment 5. The method of embodiment 1, wherein the edit site is amutation in a disease-associated gene.

Embodiment 6. The method of embodiment 5, wherein the mutation is asingle base substitution, insertion, deletion, or inversion.

Embodiment 7. The method of embodiment 1, wherein the disease-associatedgene is associated with a monogenetic disorder selected from the groupconsisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 AntitrypsinDeficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; MarfanSyndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease;Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.

Embodiment 8. The method of embodiment 1, wherein the disease-associatedgene is associated with a polygenic disorder selected from the groupconsisting of: heart disease; high blood pressure; Alzheimer's disease;arthritis; diabetes; cancer; and obesity.

Embodiment 9. The method of embodiment 1, wherein the fusion protein hasan amino acid sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277,325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or an amino acidsequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 499-505, 735-761, 776-777.

Embodiment 10. The method of embodiment 1, wherein the napDNAbp is Cas9,Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute, or avariant of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, orArgonaute.

Embodiment 11. The method of embodiment 1, wherein the napDNAbp is aCas9 or variant thereof.

Embodiment 12. The method of embodiment 1, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

Embodiment 13. The method of embodiment 1, wherein the napDNAbp is Cas9nickase (nCas9).

Embodiment 14. The method of embodiment 1, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NO: 18, or an amino acidsequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with SEQ ID NO: 18.

Embodiment 15. The method of embodiment 1, wherein the napDNAbp isSpCas9 wild type or a variant thereof of any one of amino acid sequencesSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487, or an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with any of SEQ ID NOs: 18-88, 126, 130,137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 16. The method of embodiment 1, wherein the napDNAbp is anSpCas9 ortholog.

Embodiment 17. The method of embodiment 1, wherein the napDNAbp is anyone of amino acid sequences SEQ ID NOs: 18-88, 126, 130, 137, 141, 147,153, 157, 445, 460, 467, and 482-487, or an amino acid sequence havingat least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 18. The method of embodiment 1, wherein the napDNAbpcomprises an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with the amino acid sequence of any one ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 19. The method of embodiment 1, wherein the polymerasecomprises an RNA-dependent DNA polymerase activity.

Embodiment 20. The method of embodiment 1, wherein the polymerase is areverse transcriptase.

Embodiment 21. The method of embodiment 1, wherein the reversetranscriptase is a naturally occurring wild type reverse transcriptasehaving an amino acid sequence of any one of SEQ ID NOs: 89 or an aminoacid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with any of SEQ ID NOs: 89.

Embodiment 22. The method of embodiment 1, wherein the reversetranscriptase is a variant reverse transcriptase having an amino acidsequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139,143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and766 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%,or 99% sequence identity with any of SEQ ID NOs: 89-100, 105-122,128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700,701-716, 739-741, and 766.

Embodiment 23. The method of embodiment 1, wherein the reversetranscriptase comprises any one of the amino acid sequences of SEQ IDNOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454,471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 24. The method of embodiment 1, wherein the reversetranscriptase comprises an amino acid sequence having at least 80%, 85%,90%, 95%, 98%, or 99% sequence identity with the amino acid sequence ofany one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149,154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Embodiment 25. The method of embodiment 1, wherein the detectablesequence is an insertion of at least 1, or at least 2, or at least 3, orat least 4, or at least 5, or at least 6, or at least 7, or at least 8,or at least 9, or at least 10, or at least 11, or at least 12, or atleast 13, or at least 14, or at least 15, or at least 16, or at least17, or at least 18, or at least 19, or at least 20, or at least 21, orat least 22, or at least 23, or at least 24, or at least 25, or at least26, or at least 27, or at least 28, or at least 29, or at least 30, orat least 31, or at least 32, or at least 33, or at least 34, or at least35, or at least 40, or at least 50, or at least 60, or at least 70, orat least 80, or at least 90, or at least 100 nucleobases.

Embodiment 26. The method of embodiment 1, wherein the detectablesequence is a deletion of at least 1, or at least 2, or at least 3, orat least 4, or at least 5, or at least 6, or at least 7, or at least 8,or at least 9, or at least 10, or at least 11, or at least 12, or atleast 13, or at least 14, or at least 15, or at least 16, or at least17, or at least 18, or at least 19, or at least 20, or at least 21, orat least 22, or at least 23, or at least 24, or at least 25, or at least26, or at least 27, or at least 28, or at least 29, or at least 30, orat least 31, or at least 32, or at least 33, or at least 34, or at least35, or at least 40, or at least 50, or at least 60, or at least 70, orat least 80, or at least 90, or at least 100 nucleobases.

Embodiment 27. The method of embodiment 1, wherein the detectablesequence is a nucleobase substitution.

Embodiment 28. The method of embodiment 1, wherein the detectablesequence is a transition mutation.

Embodiment 29. The method of embodiment 1, wherein the detectablesequence is a transversion mutation.

Embodiment 30. The method of embodiment 1, wherein the detectablesequence is a single nucleotide substitution selected from the groupconsisting of: (1) a G to T substitution, (2) a G to A substitution, (3)a G to C substitution, (4) a T to G substitution, (5) a T to Asubstitution, (6) a T to C substitution, (7) a C to G substitution, (8)a C to T substitution, (9) a C to A substitution, (10) an A to Tsubstitution, (11) an A to G substitution, and (12) an A to Csubstitution.

Embodiment 31. The method of embodiment 1, wherein the detectablesequence is a single nucleotide substitution that converts (1) a G:Cbasepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) aG:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5)a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair,(7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:Abasepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair toa T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:Tbasepair to a C:G basepair.

Embodiment 32. The method of embodiment 1, wherein the detectablesequence is a barcode sequence.

Embodiment 33. The method of embodiment 1, wherein step (d) ofdetermining the nucleotide sequence at the on-target and off-targetsites comprises (i) fragmenting the target nucleotide sequence to formfragments, (ii) attaching adapter sequences to the ends of thefragments, (iii) PCR amplifying an amplicon using a pair of primerswherein one primer anneals to an adapter sequence attached at one end ofthe fragments, and another primer that anneals to an adapter sequenceinserted by prime editing located within the fragment, and (iv)sequencing the amplicon to determine the location of the edit.

Group J. PE Methods for Cell Data Recording

Embodiment 1. A method of recording a cellular event by prime editing,the method comprising: (A) introducing into a cell one or moreconstructs encoding (i) a prime editor fusion protein comprising annapDNAbp and an RNA-dependent DNA polymerase and (ii) a PEgRNA, whereinthe expression of the fusion protein and/or the PEgRNA is induced by theoccurrence of a cellular event, and wherein upon expression of thefusion protein and/or the PEgRNA results in prime editing of a targetedit site in the genome of the cell to introduce a detectable sequence,and (B) identifying the detectable sequence, thereby identifying theoccurrence of the cellular event.

Embodiment 2. The method of embodiment 1, wherein the prime editing ofstep (A) simultaneously introduces a new target edit site such that therecording of the cellular event may occur iteratively.

Embodiment 3. The method of embodiment 1, wherein the PEgRNA comprisesan edit template that encodes the detectable sequence.

Embodiment 4. The method of embodiment 3, wherein the edit templatefurther encodes a new target edit site.

Embodiment 5. The method of embodiment 1, wherein the detectablesequence is an insertion of at least 1, or at least 2, or at least 3, orat least 4, or at least 5, or at least 6, or at least 7, or at least 8,or at least 9, or at least 10, or at least 11, or at least 12, or atleast 13, or at least 14, or at least 15, or at least 16, or at least17, or at least 18, or at least 19, or at least 20, or at least 21, orat least 22, or at least 23, or at least 24, or at least 25, or at least26, or at least 27, or at least 28, or at least 29, or at least 30, orat least 31, or at least 32, or at least 33, or at least 34, or at least35, or at least 40, or at least 50, or at least 60, or at least 70, orat least 80, or at least 90, or at least 100 nucleobases.

Embodiment 6. The method of embodiment 1, wherein the detectablesequence is a deletion of at least 1, or at least 2, or at least 3, orat least 4, or at least 5, or at least 6, or at least 7, or at least 8,or at least 9, or at least 10, or at least 11, or at least 12, or atleast 13, or at least 14, or at least 15, or at least 16, or at least17, or at least 18, or at least 19, or at least 20, or at least 21, orat least 22, or at least 23, or at least 24, or at least 25, or at least26, or at least 27, or at least 28, or at least 29, or at least 30, orat least 31, or at least 32, or at least 33, or at least 34, or at least35, or at least 40, or at least 50, or at least 60, or at least 70, orat least 80, or at least 90, or at least 100 nucleobases.

Embodiment 7. The method of embodiment 1, wherein the detectablesequence is a nucleobase substitution.

Embodiment 8. The method of embodiment 1, wherein the detectablesequence is a transition mutation.

Embodiment 9. The method of embodiment 1, wherein the detectablesequence is a transversion mutation.

Embodiment 10. The method of embodiment 1, wherein the detectablesequence is a single nucleotide substitution of wherein the singlenucleotide substitution is (1) a G to T substitution, (2) a G to Asubstitution, (3) a G to C substitution, (4) a T to G substitution, (5)a T to A substitution, (6) a T to C substitution, (7) a C to Gsubstitution, (8) a C to T substitution, (9) a C to A substitution, (10)an A to T substitution, (11) an A to G substitution, or (12) an A to Csubstitution.

Embodiment 11. The method of embodiment 1, wherein the detectablesequence is a single nucleotide substitution that converts (1) a G:Cbasepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) aG:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5)a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair,(7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:Abasepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair toa T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:Tbasepair to a C:G basepair.

Embodiment 12. The method of embodiment 1, wherein the detectablesequence is a barcode sequence.

Embodiment 13. The method of embodiment 1, wherein the detectablesequence increases in length over time as a result of iterativeinsertion of the detectable sequence for each occurrence of the cellularevent.

Embodiment 14. The method of embodiment 1, wherein the detecting stepcomprises sequencing the edited target site, or an amplicon of theedited target site.

Embodiment 15. The method of embodiment 1, wherein the napDNAbp is Cas9,Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute, or avariant of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, orArgonaute.

Embodiment 16. The method of embodiment 1, wherein the napDNAbp is aCas9 or variant thereof.

Embodiment 17. The method of embodiment 1, wherein the napDNAbp is anuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9nickase (nCas9).

Embodiment 18. The method of embodiment 1, wherein the napDNAbp is Cas9nickase (nCas9).

Embodiment 19. The method of embodiment 1, wherein the napDNAbpcomprises the amino acid sequence of SEQ ID NO: 18, or an amino acidsequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with SEQ ID NO: 18.

Embodiment 20. The method of embodiment 1, wherein the napDNAbp isSpCas9 wild type or a variant thereof of any one of amino acid sequencesSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487, or an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity with any of SEQ ID NOs: 18-88, 126, 130,137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

Embodiment 21. The method of embodiment 1, wherein the napDNAbp is anSpCas9 ortholog.

Embodiment 22. The method of embodiment 1, wherein the napDNAbp is anyone of amino acid sequences SEQ ID NOs: 18-88, 126, 130, 137, 141, 147,153, 157, 445, 460, 467, and 482-487, or an amino acid sequence havingat least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any ofSEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 23. The method of embodiment 1, wherein the RNA-dependent DNApolymerase is a reverse transcriptase.

Embodiment 24. The method of embodiment 23, wherein the reversetranscriptase is a naturally occurring wild type reverse transcriptasehaving an amino acid sequence of any one of SEQ ID NOs: 89 or an aminoacid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with any of SEQ ID NOs: 89.

Embodiment 25. The method of embodiment 23, wherein the reversetranscriptase is a variant reverse transcriptase having an amino acidsequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139,143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and766 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%,or 99% sequence identity with any of SEQ ID NOs: 89-100, 105-122,128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700,701-716, 739-741, and 766.

Embodiment 26. The method of embodiment 1, wherein the fusion proteincomprises an amino acid sequence of any one of SEQ ID NOs: 123 and 134(PE1, PE2), or an amino acid sequence having at least 80%, 85%, 90%,95%, 98%, or 99% sequence identity with any of SEQ ID NOs:123 and 134(PE1, PE2).

Embodiment 27. The method of embodiment 1, wherein the one or moreconstructs that encode the prime editor fusion protein and/or the PEgRNAfurther comprises one or more promoters which are inducible whencellular event occurs.

Embodiment 28. The method of embodiment 1, wherein the cellular event ismarked by a stimulus received by the cell.

Embodiment 29. The method of embodiment 28, wherein the stimulus is asmall molecule, a protein, a peptide, an amino acid, a metabolite, aninorganic molecule, an organometallic molecule, an organic molecule, adrug or drug candidate, a sugar, a lipid, a metal, a nucleic acid, amolecule produced during the activation of an endogenous or an exogenoussignaling cascade, light, heat, sound, pressure, mechanical stress,shear stress, or a virus or other microorganism, change in pH, or changein oxidation/reduction state.

Embodiment 30. A cell data recording plasmid for recording a cellularevent using prime editing, comprising:

-   -   i. a fusion protein comprising (a) a nucleic acid sequence        encoding a nucleic acid programmable DNA binding protein        (napDNAbp) and a (b) RNA-dependent DNA polymerase, said fusion        protein being operably linked to a first promoter;    -   ii. a nucleic acid sequence encoding a prime editor guide RNA        (PEgRNA) operably linked to a second promoter, wherein the        PEgRNA is complementary to a target sequence; and    -   iii. an origin of replication;    -   wherein at least one of the promoters is an inducible promoter,        and wherein the PEgRNA associates with the napDNAbp under        conditions that induce expression of the PEgRNA and expression        of the napDNAbp sufficiently to install a detectable sequence at        a target edit site.

Embodiment 31. The cell data recording plasmid of embodiment 30, whereinthe inducible promoter is induced by the cellular event.

Embodiment 32. The cell data recording plasmid of embodiment 30, whereinthe cellular event is marked by a stimulus received by the cell.

Embodiment 33. The cell data recording plasmid of embodiment 32, whereinthe stimulus is a small molecule, a protein, a peptide, an amino acid, ametabolite, an inorganic molecule, an organometallic molecule, anorganic molecule, a drug or drug candidate, a sugar, a lipid, a metal, anucleic acid, a molecule produced during the activation of an endogenousor an exogenous signaling cascade, light, heat, sound, pressure,mechanical stress, shear stress, or a virus or other microorganism,change in pH, or change in oxidation/reduction state.

Embodiment 34. The cell data recording plasmid of embodiment 30, whereinthe first and second promoter are the same.

Embodiment 35. The cell data recording plasmid of embodiment 30, whereinthe first and second promoter are different.

Embodiment 36. The cell data recording plasmid of embodiment 30, whereinthe at least one inducible promoter is an anhydrotetracycline-induciblepromoter, IPTG-inducible promoter, rhamnose-inducible promoter, orarabinose-inducible promoter.

Embodiment 37. The cell data recording plasmid of embodiment 30, whereinthe first or second promoter is a constitutive promoter.

Embodiment 38. The cell data recording plasmid of embodiment 37, whereinthe constitutive promoter is a Lac promoter, cytomegalovirus (CMV)promoter, a constitutive RNA polymerase III promoter, or a UBC promoter.

Embodiment 39. The cell data recording plasmid of embodiment 30, whereinthe origin of replication comprises a pSC101, pMB1, pBR322, ColE1, orp15A origin of replication sequence.

Embodiment 40. The cell data recording plasmid of embodiment 30, whereinthe PEgRNA comprises an edit template that encodes the detectablesequence.

Embodiment 41. The cell data recording plasmid of embodiment 40, whereinthe edit template further encodes a new target edit site.

Embodiment 42. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence is an insertion of at least 1, or at least 2, orat least 3, or at least 4, or at least 5, or at least 6, or at least 7,or at least 8, or at least 9, or at least 10, or at least 11, or atleast 12, or at least 13, or at least 14, or at least 15, or at least16, or at least 17, or at least 18, or at least 19, or at least 20, orat least 21, or at least 22, or at least 23, or at least 24, or at least25, or at least 26, or at least 27, or at least 28, or at least 29, orat least 30, or at least 31, or at least 32, or at least 33, or at least34, or at least 35, or at least 40, or at least 50, or at least 60, orat least 70, or at least 80, or at least 90, or at least 100nucleobases.

Embodiment 43. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence is a deletion of at least 1, or at least 2, orat least 3, or at least 4, or at least 5, or at least 6, or at least 7,or at least 8, or at least 9, or at least 10, or at least 11, or atleast 12, or at least 13, or at least 14, or at least 15, or at least16, or at least 17, or at least 18, or at least 19, or at least 20, orat least 21, or at least 22, or at least 23, or at least 24, or at least25, or at least 26, or at least 27, or at least 28, or at least 29, orat least 30, or at least 31, or at least 32, or at least 33, or at least34, or at least 35, or at least 40, or at least 50, or at least 60, orat least 70, or at least 80, or at least 90, or at least 100nucleobases.

Embodiment 44. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence is a nucleobase substitution.

Embodiment 45. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence is a transition mutation.

Embodiment 46. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence is a transversion mutation.

Embodiment 47. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence is a single nucleotide substitution of whereinthe single nucleotide substitution is (1) a G to T substitution, (2) a Gto A substitution, (3) a G to C substitution, (4) a T to G substitution,(5) a T to A substitution, (6) a T to C substitution, (7) a C to Gsubstitution, (8) a C to T substitution, (9) a C to A substitution, (10)an A to T substitution, (11) an A to G substitution, or (12) an A to Csubstitution.

Embodiment 48. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence is a single nucleotide substitution thatconverts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to anA:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair toa G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:Abasepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) aC:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair,(10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:Cbasepair, or (12) an A:T basepair to a C:G basepair.

Embodiment 49. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence is a barcode sequence.

Embodiment 50. The cell data recording plasmid of embodiment 30, whereinthe detectable sequence increases in length over time as a result ofiterative insertion of the detectable sequence for each occurrence ofthe cellular event.

Embodiment 51. The cell data recording plasmid of embodiment 30, whereinthe detecting step comprises sequencing the edited target site, or anamplicon of the edited target site.

Embodiment 52. The cell data recording plasmid of embodiment 30, whereinthe napDNAbp is Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c,or Argonaute, or a variant of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1,Cas13a, Cas12c, or Argonaute.

Embodiment 53. The cell data recording plasmid of embodiment 30, whereinthe napDNAbp is a Cas9 or variant thereof.

Embodiment 54. The cell data recording plasmid of embodiment 30, whereinthe napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9(dCas9), or a Cas9 nickase (nCas9).

Embodiment 55. The cell data recording plasmid of embodiment 30, whereinthe napDNAbp is Cas9 nickase (nCas9).

Embodiment 56. The cell data recording plasmid of embodiment 30, whereinthe napDNAbp comprises the amino acid sequence of SEQ ID NO: 18, or anamino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity with SEQ ID NO: 18.

Embodiment 57. The cell data recording plasmid of embodiment 30, whereinthe napDNAbp is SpCas9 wild type or a variant thereof of any one ofamino acid sequences SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487, or an amino acid sequence having atleast 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and482-487.

Embodiment 58. The cell data recording plasmid of embodiment 30, whereinthe napDNAbp is an SpCas9 ortholog.

Embodiment 59. The cell data recording plasmid of embodiment 30, whereinthe napDNAbp is any one of amino acid sequences SEQ ID NOs: 18-88, 126,130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487, or an aminoacid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153,157, 445, 460, 467, and 482-487.

Embodiment 60. The cell data recording plasmid of embodiment 30, whereinthe RNA-dependent DNA polymerase is a reverse transcriptase.

Embodiment 61. The cell data recording plasmid of embodiment 30, whereinthe reverse transcriptase is a naturally occurring wild type reversetranscriptase having an amino acid sequence of any one of SEQ ID NOs: 89or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or99% sequence identity with any of SEQ ID NOs: 89.

Embodiment 62. The cell data recording plasmid of embodiment 30, whereinthe reverse transcriptase is a variant reverse transcriptase having anamino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129,132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716,739-741, and 766 or an amino acid sequence having at least 80%, 85%,90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 89-100,105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662,700, 701-716, 739-741, and 766.

Embodiment 63. The cell data recording plasmid of embodiment 30, whereinthe fusion protein comprises an amino acid sequence of any one of SEQ IDNOs: 123 and 134 (PE1, PE2), or an amino acid sequence having at least80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ IDNOs: 123 and 134 (PE1, PE2).

Embodiment 64. A kit for use in a cell comprising the cell data recorderplasmid of any one of embodiments 30 63.

Embodiment 65. The kit of embodiment 64, wherein the cell is aprokaryotic cell.

Embodiment 66. The kit of embodiment 64, wherein the cell is aeukaryotic cell.

Embodiment 67. A cell comprising the cell data recording plasmid of anyone of embodiments 30-63.

Embodiment 68. The cell of embodiment 67, wherein the cell is aprokaryotic cell.

Embodiment 69. The cell of embodiment 67, wherein the cell is aeukaryotic cell.

Embodiment 70. The cell of embodiment 69, wherein the eukaryotic cell isa mammalian cell.

Embodiment 71. The cell of embodiment 70, wherein the mammalian cell isa human cell.

Embodiment 72. A method of recording a cellular event by prime editing,the method comprising: (A) introducing into a cell a cell data recordingplasmid of any of embodiments 30-63, wherein the fusion protein and/orthe PEgRNA are induced by the occurrence of a cellular event, andwherein expression of the fusion protein and/or the PEgRNA results inprime editing of a target edit site in the genome of the cell tointroduce a detectable sequence, and (B) identifying the detectablesequence, thereby identifying the occurrence of the cellular event.

Embodiment 73. The method of embodiment 72, wherein the step of (A)introducing is by transfection or electroporation.

EQUIVALENTS AND SCOPE

In the articles such as “a,” “an,” and “the” may mean one or more thanone unless indicated to the contrary or otherwise evident from thecontext. Embodiments or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the disclosure encompasses all variations, combinations,and permutations in which one or more limitations, elements, clauses,and descriptive terms from one or more of the listed claims isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claims that is dependent on the same base claim.Where elements are presented as lists, e.g., in Markush group format,each subgroup of the elements is also disclosed, and any element(s) canbe removed from the group. It should it be understood that, in general,where the invention, or aspects of the invention, is/are referred to ascomprising particular elements and/or features, certain embodiments ofthe disclosure or aspects of the disclosure consist, or consistessentially of, such elements and/or features. For purposes ofsimplicity, those embodiments have not been specifically set forth inhaec verba herein. It is also noted that the terms “comprising” and“containing” are intended to be open and permits the inclusion ofadditional elements or steps. Where ranges are given, endpoints areincluded. Furthermore, unless otherwise indicated or otherwise evidentfrom the context and understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value orsub-range within the stated ranges in different embodiments of theinvention, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the embodiments. Because suchembodiments are deemed to be known to one of ordinary skill in the art,they may be excluded even if the exclusion is not set forth explicitlyherein. Any particular embodiment of the invention can be excluded fromany embodiment, for any reason, whether or not related to the existenceof prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended embodiments. Those ofordinary skill in the art will appreciate that various changes andmodifications to this description may be made without departing from thespirit or scope of the present invention, as defined in the followingembodiments.

1-51. (canceled)
 52. A prime editing system comprising: (a) a primeediting guide RNA (PEgRNA), or one or more polynucleotides encoding thePEgRNA, wherein the PEgRNA comprises: (i) a spacer sequence comprising aregion of complementarity to a first strand of a double-stranded DNAsequence; (ii) a gRNA core capable of complexing with a nucleic acidprogrammable DNA binding protein (napDNAbp) that is configured togenerate a cut site in a second strand of the double-stranded DNAsequence; and (iii) an extension arm comprising: A. a DNA synthesistemplate encoding a recombinase recognition sequence or its reversecomplement, and B. a primer binding site that is complementary to aregion upstream of the cut site in the second strand of thedouble-stranded DNA sequence; (b) a prime editor, or one or morepolynucleotides encoding the prime editor, the prime editor comprising:(i) the nucleic acid programmable DNA binding protein (napDNAbp) that isconfigured to generate the cut site in a second strand of thedouble-stranded DNA sequence; and (ii) a DNA polymerase domain; and (c)a recombinase that recognizes the recombinase recognition sequence orits reverse complement, or one or more polynucleotides encoding therecombinase.
 53. The prime editing system of claim 52 further comprisinga donor template or a polynucleotide encoding the donor template. 54.The prime editing system of claim 52, wherein the recombinaserecognition sequence or its reverse complement is recognized by arecombinase selected from the group consisting of serine recombinases,tyrosine recombinases, resolvases, invertases, integrases, serineintegrases, tyrosine integrases, phage integrases, and transposases. 55.The prime editing system of claim 52, wherein the recombinaserecognition sequence or its reverse complement is recognized by Bxb1.56. The prime editing system of claim 52, wherein the recombinaserecognition sequence or its reverse complement is SEQ ID NO: 537 or 536.57. The prime editing system of claim 56, further comprising a donortemplate, wherein: (a) the donor template comprises SEQ ID NO: 536 andthe recombinase recognition sequence or its reverse complement is SEQ IDNO: 537; or (b) the donor template comprises SEQ ID NO: 537 and therecombinase recognition sequence or its reverse complement is SEQ ID NO:536.
 58. The prime editing system of claim 52, wherein the extension armencodes two recombinase recognition sequences or their reversecomplements.
 59. The prime editing system of claim 52, wherein the DNAsynthesis template further comprises a homology arm that iscomplementary to a region downstream of the cut site in the secondstrand of the double-stranded DNA sequence.
 60. The prime editing systemof claim 59, wherein the homology arm is from 5 to 20 nucleotides inlength.
 61. The prime editing system of claim 59, wherein the homologyarm is located 5′ of the recombinase recognition sequence or its reversecomplement in the DNA synthesis template.
 62. The prime editing systemof claim 52, wherein the DNA synthesis template encodes a sequence thatwhen integrated into the double-stranded DNA sequence disrupts anendogenous PAM site associated with the spacer sequence.
 63. The primeediting system of claim 62, wherein the recombinase recognition sequenceor its reverse complement is 5′ of and directly adjacent to the primerbinding site.
 64. The prime editing system of claim 59, wherein theextension arm comprises, from 5′ to 3′, the homology arm, therecombinase recognition sequence or its reverse complement, and theprimer binding site.
 65. The prime editing system of claim 64, whereinthe recombinase recognition sequence or its reverse complement isdirectly adjacent to the primer binding site.
 66. The prime editingsystem of claim 52, wherein the region upstream of the cut site to whichprimer binding site is complementary is immediately 5′ of the cut site.67. The prime editing system of claim 52, wherein the cut site is threenucleotides upstream of a protospacer adjacent motif (PAM) in the secondstrand of the double-stranded DNA sequence.
 68. The prime editing systemof claim 52, wherein the primer binding site is from 7 to 17 nucleotidesin length.
 69. The prime editing system of claim 52, wherein the primerbinding site is from 8 to 15 nucleotides in length.
 70. The primeediting system of claim 52, wherein the spacer sequence is 20nucleotides in length.
 71. The prime editing system of claim 52, whereinthe PEgRNA is a single molecule comprising in a 5′ to 3′ orientation:the spacer sequence, the gRNA core, and the extension arm.
 72. The primeediting system of claim 52, wherein the PEgRNA comprises at least oneof: a modified nucleobase, a modified sugar, a modified phosphate group,or a nucleoside analog.
 73. The prime editing system of claim 52,wherein the PEgRNA comprises one or more 3′ structures selected from thegroup consisting of linkers, stem loops, hairpins, toeloops, tetraloops,aptamers, and RNA-protein recruitment domains.
 74. The prime editingsystem of claim 52, wherein the PEgRNA comprises an aptamer capable ofrecruiting an effector domain, optionally where the aptamer is an MS2aptamer.
 75. The prime editing system of claim 52, wherein the nucleicacid programmable DNA binding protein is a CRISPR-Cas effector protein.76. The prime editing system of claim 52, wherein the nucleic acidprogrammable DNA binding protein comprises an RuvC endonuclease domain,an HNH endonuclease domain, or both.
 77. The prime editing system ofclaim 76, wherein the nucleic acid programmable DNA binding proteincomprises an HNH endonuclease domain and a RuvC endonuclease domain, andwherein the HNH endonuclease domain comprises one or more mutations thatdecrease or eliminate nuclease activity.
 78. The prime editing system ofclaim 52, wherein the nucleic acid programmable DNA binding protein is aCas9 nickase or a Cas9 nuclease.
 79. The prime editing system of claim52, wherein the extension arm is an RNA extension arm. and wherein theDNA polymerase domain is a reverse transcriptase domain.
 80. The primeediting system of claim 79, wherein the reverse transcriptase domain isderived from a retrovirus or a retrotransposon.
 81. The prime editingsystem of claim 79, wherein the reverse transcriptase domain is aMoloney-Murine Leukemia Virus reverse transcriptase (M-MLV RT).